Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts.

BACKGROUND: Myelodysplastic syndromes are a diverse and common group of chronic hematologic cancers. The identification of new genetic lesions could facilitate new diagnostic and therapeutic strategies. METHODS: We used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 9 patients with low-grade myelodysplasia. Targeted resequencing of the gene encoding RNA splicing factor 3B, subunit 1 (SF3B1), was also performed in a cohort of 2087 patients with myeloid or other cancers. RESULTS: We identified 64 point mutations in the 9 patients. Recurrent somatically acquired mutations were identified in SF3B1. Follow-up revealed SF3B1 mutations in 72 of 354 patients (20%) with myelodysplastic syndromes, with particularly high frequency among patients whose disease was characterized by ring sideroblasts (53 of 82 [65%]). The gene was also mutated in 1 to 5% of patients with a variety of other tumor types. The observed mutations were less deleterious than was expected on the basis of chance, suggesting that the mutated protein retains structural integrity with altered function. SF3B1 mutations were associated with down-regulation of key gene networks, including core mitochondrial pathways. Clinically, patients with SF3B1 mutations had fewer cytopenias and longer event-free survival than patients without SF3B1 mutations. CONCLUSIONS: Mutations in SF3B1 implicate abnormalities of messenger RNA splicing in the pathogenesis of myelodysplastic syndromes. (Funded by the Wellcome Trust and others.).

Report of consensus panel 7 from the 11th international workshop on Waldenström macroglobulinemia on priorities for novel clinical trials.

Recent advances in the understanding of Waldenström macroglobulinemia (WM) biology have impacted the development of effective novel agents and improved our knowledge of how the genomic background of WM may influence selection of therapy. Consensus Panel 7 (CP7) of the 11th International Workshop on WM was convened to examine the current generation of completed and ongoing clinical trials involving novel agents, consider updated data on WM genomics, and make recommendations on the design and prioritization of future clinical trials. CP7 considers limited duration and novel-novel agent combinations to be the priority for the next generation of clinical trials. Evaluation of MYD88, CXCR4 and TP53 at baseline in the context of clinical trials is crucial. The common chemoimmunotherapy backbones, bendamustine-rituximab (BR) and dexamethasone, rituximab and cyclophosphamide (DRC), may be considered standard-of-care for the frontline comparative studies. Key unanswered questions include the definition of frailty in WM; the importance of attaining a very good partial response or better (≥VGPR), within stipulated time frame, in determining survival outcomes; and the optimal treatment of WM populations with special needs.

The roles of homologous recombination and the immune system in the genomic evolution of cancer.

A variety of factors, whether extracellular (mutagens/carcinogens and viruses in the environment, chronic inflammation and radiation associated with the environment and/or electronic devices/machines) and/or intracellular (oxidative metabolites of food, oxidative stress due to inflammation, acid production, replication stress, DNA replication/repair errors, and certain hormones, cytokines, growth factors), pose a constant threat to the genomic integrity of a living cell. However, in the normal cellular environment multiple biological pathways including DNA repair, cell cycle, apoptosis and the immune system work in a precise, regulated (tightly controlled), timely and concerted manner to ensure genomic integrity, stability and proper functioning of a cell. If damage to DNA takes place, it is efficiently and accurately repaired by the DNA repair systems. Homologous recombination (HR) which utilizes either a homologous chromosome (in G1 phase) or a sister chromatid (in G2) as a template to repair the damage, is known to be the most precise repair system. HR in G2 which utilizes a sister chromatid as a template is also called an error free repair system. If DNA damage in a cell is so extensive that it overwhelms the repair system/s, the cell is eliminated by apoptosis. Thus, multiple pathways ensure that genome of a cell is intact and stable. However, constant exposure to DNA damage and/or dysregulation of DNA repair mechanism/s poses a risk of mutation and cancer. Oncogenesis, which seems to be a multistep process, is associated with acquisition of a number of genomic changes that enable a normal cell to progress from benign to malignant transformation. Transformed/cancer cells are recognized and killed by the immune system. However, the ongoing acquisition of new genomic changes enables cancer cells to survive/escape immune attack, evolve into a more aggressive phenotype, and eventually develop resistance to therapy. Although DNA repair (especially the HR) and the immune system play unique roles in preserving genomic integrity of a cell, they can also contribute to DNA damage, genomic instability and oncogenesis. The purpose of this article is to highlight the roles of DNA repair (especially HR) and the immune system in genomic evolution, with special focus on gastrointestinal cancer.

Identification and characterization of HLA-A24-specific XBP1, CD138 (Syndecan-1) and CS1 (SLAMF7) peptides inducing antigens-specific memory cytotoxic T lymphocytes targeting multiple myeloma.

X-box binding protein 1 (XBP1), CD138 (Syndecan-1) and CS1 (SLAMF7) are highly expressed antigens in cancers including multiple myeloma (MM). Here, we identify and characterize immunogenic HLA-A24 peptides derived from these antigens for potential vaccination therapy of HLA-A24+ patients with MM. The identified immunogenic HLA-A24-specific XBP1 unspliced (UN)185-193 (I S P W I L A V L), XBP1 spliced (SP)223-231 (V Y P E G P S S L), CD138265-273 (I F A V C L V G F) and CS1240-248 (L F V L G L F L W) peptides induced antigen-specific CTL with anti-MM activity in an HLA-A24 restricted manner. Furthermore, a cocktail containing the four HLA-A24 peptides evoked MM-specific CTL with distinct phenotypic profiles (CD28, CD40L, 41BB, CD38, CD69) and anti-tumor activities, evidenced by perforin upregulation, CD107a degranulation (cytotoxicity) and Th1-type cytokines (IFN-γ/IL-2/TNF-α) production in response to HLA-A24+ MM cells. The multipeptide-specific CTL included antigen-specific memory CD8+ T cells expressing both T-cell activation (CD38, CD69) and immune checkpoints antigens (CTLA, PD-1, LAG-3, TIM-3). These results provide the framework for a multipeptide vaccination therapy to induce tumor-specific CTL in HLA-A24-positive patients with myeloma and other cancers expressing these antigens.

Protein arginine methyltransferase 5 has prognostic relevance and is a druggable target in multiple myeloma.

Arginine methyltransferases critically regulate cellular homeostasis by modulating the functional outcome of their substrates. The protein arginine methyltransferase 5 (PRMT5) is an enzyme involved in growth and survival pathways promoting tumorigenesis. However, little is known about the biologic function of PRMT5 and its therapeutic potential in multiple myeloma (MM). In the present study, we identified and validated PRMT5 as a new therapeutic target in MM. PRMT5 is overexpressed in patient MM cells and associated with decreased progression-free survival and overall survival. Either genetic knockdown or pharmacological inhibition of PRMT5 with the inhibitor EPZ015666 significantly inhibited growth of both cell lines and patient MM cells. Furthermore, PRMT5 inhibition abrogated NF-κB signaling. Interestingly, mass spectrometry identified a tripartite motif-containing protein 21 TRIM21 as a new PRMT5-partner, and we delineated a TRIM21-dependent mechanism of NF-κB inhibition. Importantly, oral administration of EPZ015666 significantly decreased MM growth in a humanized murine model of MM. These data both demonstrate the oncogenic role and prognostic relevance of PRMT5 in MM pathogenesis, and provide the rationale for novel therapies targeting PRMT5 to improve patient outcome.

MiR-29b antagonizes the pro-inflammatory tumor-promoting activity of multiple myeloma-educated dendritic cells.

Dendritic cells (DCs) have a key role in regulating tumor immunity, tumor cell growth and drug resistance. We hypothesized that multiple myeloma (MM) cells might recruit and reprogram DCs to a tumor-permissive phenotype by changes within their microRNA (miRNA) network. By analyzing six different miRNA-profiling data sets, miR-29b was identified as the only miRNA upregulated in normal mature DCs and significantly downregulated in tumor-associated DCs. This finding was validated in primary DCs co-cultured in vitro with MM cell lines and in primary bone marrow DCs from MM patients. In DCs co-cultured with MM cells, enforced expression of miR-29b counteracted pro-inflammatory pathways, including signal transducer and activator of transcription 3 and nuclear factor-κB, and cytokine/chemokine signaling networks, which correlated with patients' adverse prognosis and development of bone disease. Moreover, miR-29b downregulated interleukin-23 in vitro and in the SCID-synth-hu in vivo model, and antagonized a Th17 inflammatory response. All together, these effects translated into strong anti-proliferative activity and reduction of genomic instability of MM cells. Our study demonstrates that MM reprograms the DCs functional phenotype by downregulating miR-29b whose reconstitution impairs DCs ability to sustain MM cell growth and survival. These results underscore miR-29b as an innovative and attractive candidate for miRNA-based immune therapy of MM.

Nucleotide excision repair is a potential therapeutic target in multiple myeloma.

Despite the development of novel drugs, alkylating agents remain an important component of therapy in multiple myeloma (MM). DNA repair processes contribute towards sensitivity to alkylating agents and therefore we here evaluate the role of nucleotide excision repair (NER), which is involved in the removal of bulky adducts and DNA crosslinks in MM. We first evaluated NER activity using a novel functional assay and observed a heterogeneous NER efficiency in MM cell lines and patient samples. Using next-generation sequencing data, we identified that expression of the canonical NER gene, excision repair cross-complementation group 3 (ERCC3), significantly impacted the outcome in newly diagnosed MM patients treated with alkylating agents. Next, using small RNA interference, stable knockdown and overexpression, and small-molecule inhibitors targeting xeroderma pigmentosum complementation group B (XPB), the DNA helicase encoded by ERCC3, we demonstrate that NER inhibition significantly increases sensitivity and overcomes resistance to alkylating agents in MM. Moreover, inhibiting XPB leads to the dual inhibition of NER and transcription and is particularly efficient in myeloma cells. Altogether, we show that NER impacts alkylating agents sensitivity in myeloma cells and identify ERCC3 as a potential therapeutic target in MM.

Growth factor-dependent initiation of DNA replication in nuclei isolated from an interleukin 3-dependent murine myeloid cell line.

To study the proliferative response of hematopoietic cells to growth factors at the molecular level, we developed a cell-free system for growth factor-dependent initiation of genomic DNA replication. Nuclei were isolated from the IL-3-dependent cell line NFS/N1-H7 after a 10-h period of IL-3 deprivation. Cytosolic and membrane-containing subcellular fractions were prepared from proliferating NFS/N1-H7 cells. Nuclei from the nonproliferating cells (+/- IL-3) showed essentially no incorporation of [3H]thymidine during a 16-h incubation with a mixture of unlabeled GTP, ATP, UTP, CTP, dGTP, dATP, dCTP, and [3H]dTTP. When the combination of IL-3, a cytosolic fraction, and a membrane-containing fraction from proliferating cells was added to nuclei from nonproliferating cells, a burst of [3H]thymidine incorporation into DNA began after a 12-h lag period, attained a maximal rate at 16 h, and reached a level of 860 pmol thymidine/10(6) nuclei at 24 h (corresponding to replication of approximately 56% total mouse genomic DNA). This DNA synthesis was inhibited approximately 90% by the specific DNA polymerase alpha inhibitor aphidicolin. Deletion of a single cellular component or IL-3 from the system resulted in a marked reduction of DNA replication (-membrane, 80 +/- 4%; -cytosol, 90% +/- 4%; -IL-3, 74 +/- 7% inhibition). This model requires a growth factor (IL-3), a sedimentable cell fraction containing its receptor and possibly additional membrane-associated components, and a cytosolic fraction. It appears to recapitulate the molecular events required for progression from early G1 to S phase of the cell cycle induced by IL-3 binding to its receptor.

Antimyeloma activity of two novel N-substituted and tetraflourinated thalidomide analogs.

Thalidomide alone or in combination with steroids has significant activity in multiple myeloma (MM). However, given its teratogenic potential, analogs have been synthesized, retaining the anti-MM activity without these side effects. We examined the anti-MM activity of two thalidomide analogs, CPS11 and CPS49. Direct cytotoxicity of the drugs on myeloma cell lines and patient myeloma cells was examined using thymidine uptake. Tumor cell apoptosis was evaluated by flow cytometry as well as Western blotting for caspase and PARP cleavage. Cellular signaling events were examined by immunoblotting for phosphorylated proteins. Both drugs inhibit proliferation of several MM cell lines sensitive and resistant to conventional therapies. They decrease secretion of IL-6, IGF, and VEGF by marrow stromal cells. Importantly, they inhibit proliferation of MM cells adherent to stromal cells. These drugs induce caspase-mediated apoptosis in MM cell lines, as well as patient MM cells. They inhibit the PI3K/Akt and JAK/STAT (signal transducers and activators of transcription) pathways in MM cells and are antiangiogenic in matrigel-based assays. CPS11 and CPS49 have potent antimyeloma activity and can overcome protective effects of the tumor microenvironment. They have potent antiangiogenic activity and direct effect on bone marrow stroma. These encouraging preclinical data provide the basis for further evaluation in the clinic.

Effect of tumor irradiation on the uptake of lymphokine-activated killer cells in a murine tumor model.

Immunotherapy with lymphokine-activated killer (LAK) cells and interleukin 2 is one of the newer treatment modalities for cancer. This raises important questions concerning synergism or suppressive effects of other existing treatment modalities on adoptive immunotherapy with LAK cells. A tumor model with H4IIe hepatoma cells grown on each flank of ACI rats was developed to evaluate the effect of external beam irradiation of tumors on the subsequent concentration of LAK cells in these tumors. Tumors on one side were irradiated at 6, 12, or 16 Gy prior to injection of [3H]thymidine-labeled LAK cells. The effect of irradiation was measured as the ratio of 3H recovered in the unirradiated tumor compared to that in the irradiated tumor in the same animal as a function of dose and time after irradiation. This ratio was significantly greater than 1.0 for a radiation dose of 12 Gy (2.35 +/- 0.51) measured 2 days after irradiation, indicating a reduction in LAK cell numbers in the irradiated tumor. This reduction in LAK cell number persists up to at least 4 days following radiation exposure. A similar experiment using 125I-labeled interleukin 2 showed equal distribution in the irradiated and unirradiated tumors. Our data demonstrates that the concentration of LAK cells is markedly reduced by prior radiation, in contradistinction to increased uptake of immunoglobulins in irradiated tumors, as shown by others. If a similar reduction is observed for longer duration after radiation exposure, it might suggest a clinically important interaction between prior radiation exposure and adoptive immunotherapy.

Interaction of adeno-associated virus Rep78 with p53: implications in growth inhibition.

Adeno-associated virus (AAV) is a nonpathogenic, single-stranded DNA virus belonging to the parvoviridae family. Onco-suppressive properties of AAV against adenovirus, a DNA tumor virus, have been well documented. Rep78, a major regulatory protein of AAV, is believed to be responsible for its antioncogenic properties. Most DNA tumor viruses disturb the cell cycle pathways by essentially abrogating the functions of p53. Here we present evidence that AAV acts as an antiproliferative agent against adenovirus by protecting the adenoviral-mediated degradation of p53 as confirmed by both Western blot analysis and immunoprecipitation analysis with anti-p53 antibody. Coimmunoprecipitation experiments revealed that the AAV Rep78 is physically bound to p53 in vivo. Furthermore, the binding of purified p53 to the AAV Rep78 affinity column confirms their interaction. These results document for the first time that the antiproliferative effects of AAV against adenovirus are mediated, at least in part, by the interaction of AAV Rep78 with p53.

SAR650984 directly induces multiple myeloma cell death via lysosomal-associated and apoptotic pathways, which is further enhanced by pomalidomide.

The anti-CD38 monoclonal antibody SAR650984 (SAR) is showing promising clinical activity in treatment of relapsed and refractory multiple myeloma (MM). Besides effector-mediated antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity, we here define molecular mechanisms of SAR-directed MM cell death and enhanced anti-MM activity triggered by SAR with Pomalidomide (Pom). Without Fc-cross-linking agents or effector cells, SAR specifically induces homotypic aggregation (HA)-associated cell death in MM cells dependent on the level of cell surface CD38 expression, actin cytoskeleton and membrane lipid raft. SAR and its F(ab)'2 fragments trigger caspase 3/7-dependent apoptosis in MM cells highly expressing CD38, even with p53 mutation. Importantly, SAR specifically induces lysosome-dependent cell death (LCD) by enlarging lysosomes and increasing lysosomal membrane permeabilization associated with leakage of cathepsin B and LAMP-1, regardless of the presence of interleukin-6 or bone marrow stromal cells. Conversely, the lysosomal vacuolar H+-ATPase inhibitor blocks SAR-induced LCD. SAR further upregulates reactive oxygen species. Pom enhances SAR-induced direct and indirect killing even in MM cells resistant to Pom/Len. Taken together, SAR is the first therapeutic monoclonal antibody mediating direct cytotoxicity against MM cells via multiple mechanisms of action. Our data show that Pom augments both direct and effector cell-mediated MM cytotoxicity of SAR, providing the framework for combination clinical trials.

A DNA target-enrichment approach to detect mutations, copy number changes and immunoglobulin translocations in multiple myeloma.

Genomic lesions are not investigated during routine diagnostic workup for multiple myeloma (MM). Cytogenetic studies are performed to assess prognosis but with limited impact on therapeutic decisions. Recently, several recurrently mutated genes have been described, but their clinical value remains to be defined. Therefore, clinical-grade strategies to investigate the genomic landscape of myeloma samples are needed to integrate new and old prognostic markers. We developed a target-enrichment strategy followed by next-generation sequencing (NGS) to streamline simultaneous analysis of gene mutations, copy number changes and immunoglobulin heavy chain (IGH) translocations in MM in a high-throughput manner, and validated it in a panel of cell lines. We identified 548 likely oncogenic mutations in 182 genes. By integrating published data sets of NGS in MM, we retrieved a list of genes with significant relevance to myeloma and found that the mutational spectrum of primary samples and MM cell lines is partially overlapping. Gains and losses of chromosomes, chromosomal segments and gene loci were identified with accuracy comparable to conventional arrays, allowing identification of lesions with known prognostic significance. Furthermore, we identified IGH translocations with high positive and negative predictive value. Our approach could allow the identification of novel biomarkers with clinical relevance in myeloma.

miR-23b/SP1/c-myc forms a feed-forward loop supporting multiple myeloma cell growth.

Deregulated microRNA (miR)/transcription factor (TF)-based networks represent a hallmark of cancer. We report here a novel c-Myc/miR-23b/Sp1 feed-forward loop with a critical role in multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM) cell growth and survival. We have found miR-23b to be downregulated in MM and WM cells especially in the presence of components of the tumor bone marrow milieu. Promoter methylation is one mechanism of miR-23b suppression in myeloma. In gain-of-function studies using miR-23b mimics-transfected or in miR-23b-stably expressing MM and WM cell lines, we observed a significant decrease in cell proliferation and survival, along with induction of caspase-3/7 activity over time, thus supporting a tumor suppressor role for miR-23b. At the molecular level, miR-23b targeted Sp1 3'UTR and significantly reduced Sp1-driven nuclear factor-κB activity. Finally, c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, transcriptionally repressed miR-23b. Thus MYC-dependent miR-23b repression in myeloma cells may promote activation of oncogenic Sp1-mediated signaling, representing the first feed-forward loop with critical growth and survival role in myeloma.

Targeting IL-17A in multiple myeloma: a potential novel therapeutic approach in myeloma.

We have previously demonstrated that interleukin-17A (IL-17) producing T helper 17 cells are significantly elevated in blood and bone marrow (BM) in multiple myeloma (MM) and IL-17A promotes MM cell growth via the expression of IL-17 receptor. In this study, we evaluated anti-human IL-17A human monoclonal antibody (mAb), AIN457 in MM. We observe significant inhibition of MM cell growth by AIN457 both in the presence and the absence of BM stromal cells (BMSCs). Although IL-17A induces IL-6 production, AIN457 significantly downregulated IL-6 production and MM cell adhesion in MM-BMSC co-culture. AIN457 also significantly inhibited osteoclast cell differentiation. More importantly, in the SCIDhu model of human myeloma administration of AIN457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth and reduced bone damage compared with isotype control mice. To understand the mechanism of action of anti-IL-17A mAb, we report, here, that MM cells express IL-17A. We also observed that IL-17A knockdown inhibited MM cell growth and their ability to induce IL-6 production in co-cultures with BMSC. These pre-clinical observations suggest efficacy of AIN457 in myeloma and provide the rationale for its clinical evaluation for anti-myeloma effects and for improvement of bone disease.

Tumor antigen immunization of sibling stem cell transplant donors in multiple myeloma.

The unique antigenic determinants (idiotype (Id)) of the immunoglobulin secreted by myeloma tumor can serve as a tumor-specific antigen for active immunotherapy. Our objective was to induce tumor-specific T-cell immunity in bone marrow transplant (BMT) donors to enhance antitumor effects of allografts. We vaccinated five HLA-matched sibling donors with myeloma Id proteins isolated from recipient plasma before bone marrow harvest. Recipients were administered booster Id immunizations following transplantation. Vaccination induced donor Id and carrier-specific cellular and/or humoral immune responses. Two recipients died within 30 days of BMT from transplant-related complications. Id and carrier-specific T-cell responses were detected in all three remaining patients post-, but not pre-BMT and persisted for 18 months. All three surviving patients converted from partial to complete responses following BMT. Two of the three patients remain disease-free 7 years and 8 years after BMT, and the third died of renal failure after 5.5 years while in complete remission from myeloma. Our results suggest that myeloma Id vaccination induces specific T-cell immunity in healthy donors which may be transferable by BMT, is associated with prolonged disease-free survival of recipients, and may represent a general strategy to enhance graft-versus-tumor effect in other malignancies for which defined tumor-specific antigens exist.

Clinical activity of arsenic trioxide for the treatment of multiple myeloma.

Arsenic has been used since ancient times as a therapeutic agent. However, until recently its use in modern medicine has been restricted to the treatment of a limited number of parasitic infections. In the early 1990s, reports from China described impressive results with arsenic trioxide in patients with de novo, relapsed, and refractory acute promyelocytic leukemia (APL). Other investigators subsequently confirmed these results leading to approval of its use for relapsed or refractory APL in the United States. Investigations of this agent have demonstrated that its efficacy in APL and preclinical tumor models is dependent upon a number of mechanisms, including induction of apoptosis, effects on cellular differentiation, cell cycling, and tumor angiogenesis. Subsequent preclinical studies showed significant activity of arsenic trioxide in multiple myeloma (MM). Based on this, in a phase II trial, we have evaluated the activity of arsenic trioxide in 14 patients with relapsed MM, refractory to conventional salvage therapy. With the dose and schedule used, treatment with arsenic trioxide produced responses in three patients and prolonged stable disease in a fourth patient, with the longest response lasting 6 weeks. Although treatment was reasonably well tolerated, in these patients with extensive prior therapy, 11 developed cytopenia, five associated with infectious complications and three developed deep vein thromboses. The results of this small trial support further investigation of this novel drug for the treatment of patients with relapsed or refractory MM.

Dendritic cells derived from multiple myeloma patients efficiently internalize different classes of myeloma protein.

Myeloma protein is a unique tumor antigen that can be used to devise tumor-specific vaccination strategies. As dendritic cells (DCs) are extremely potent at inducing T-cell responses, clinical protocols have been designed using myeloma protein-pulsed DCs to elicit anti-tumor cell responses in vivo. To optimize antigen pulsing of DCs, we investigated mechanisms of antigen uptake and evaluated various laboratory parameters including class of myeloma protein, antigen exposure time, and DC maturational stage.DCs were generated by culturing peripheral blood stem cells from myeloma patients in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Myeloma proteins were labeled with fluorescein isothiocyanate (FITC) and internalization of protein by DCs was measured by flow cytometry.IgG, IgA, and free-kappa light chain myeloma proteins were all rapidly internalized by DCs in a time-dependent fashion. Maturation of DCs with tumor necrosis factor-alpha (TNF-alpha) resulted in diminished uptake. Endocytosis of myeloma protein by DCs was primarily mediated by fluid-phase macropinocytosis based on morphology, nonsaturable uptake kinetics, and sensitivity to drugs that inhibit membrane ruffling. Pulse-chase experiments revealed that the majority of internalized myeloma protein disappeared within 4 hours but was retained in the presence of chloroquine, indicating antigen processing had occurred. Cultured DCs from myeloma patients are functional and can efficiently endocytose different classes of myeloma protein by the mechanism of macropinocytosis. This demonstrates the feasibility of using all classes of myeloma protein for producing DC vaccines, and defines culture conditions for optimizing antigen loading of DCs for induction of anti-myeloma responses.

Adeno-associated virus 2-mediated gene transfer and functional expression of the human granulocyte-macrophage colony-stimulating factor.

It is becoming increasingly clear that the adeno-associated virus 2 (AAV)-based vector system may prove to be useful for high-efficiency gene transfer in human cells as well as for potential gene therapy in humans. A recombinant AAV vector containing the gene for a human hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), was constructed and used to infect COS-1 cells, a monkey kidney cell line. COS-1 cells infected with the recombinant virus, but not mock-infected cells, expressed high levels of the human GM-CSF gene transcripts. Furthermore, in co-cultivation experiments with the recombinant virus-infected cells, but not in those with mock-infected cells, active proliferation of a GM-CSF-dependent human megakaryocytic leukemia cell line, M07e, could be obtained in the absence of exogenously added GM-CSF. The recombinant GM-CSF protein released into the supernatant was biologically active in progenitor cell assays carried out with primary human hematopoietic cells, and this activity was specifically abrogated by treatment of the supernatant with anti-GM-CSF antibodies. This recombinant virus may be potentially useful in the management and gene therapy of a variety of malignant disorders in the human hematopoietic system.

CSNK1α1 mediates malignant plasma cell survival.

Here we report that targeting casein kinase 1-α1 (CSNK1α1) is a potential novel treatment strategy in multiple myeloma (MM) therapy distinct from proteasome inhibition. CSNK1α1 is expressed in all the tested MM cell lines and patient MM cells, and is not altered during bortezomib-triggered cytotoxicity. Inhibition of CSNK1α1 kinase activity in MM cells with targeted therapy D4476 or small hairpin RNAs triggers cell G0/G1-phase arrest, prolonged G2/M phase and apoptosis. D4476 also induced cytotoxicity in bortezomib-resistant MM cells and enhanced bortezomib-triggered cytotoxicity. CSNK1α1 signaling pathways include CDKN1B, P53 and FADD; gene signatures involved included interferon-α, tumor necrosis factor-α and LIN9. In addition, reduction of Csnk1α1 prevents cMYC/KRAS12V transformation of BaF3 cells independent of interleukin-3. Impartially, reducing Csnk1α1 prevented development of cMYC/KRAS12V-induced plasmacytomas in mice, suggesting that CSNK1α1 may be involved in MM initiation and progression. Our data suggest that targeting CSNK1α1, alone or combined with bortezomib, is a potential novel therapeutic strategy in MM. Moreover, inhibition of CSNK1α1 may prevent the progression of monoclonal gammopathy of undetermined significance to MM.