Myeloma Stem Cells Research Articles

Abstract LB-025: The role of NOTCH pathway in multiple myeloma associated drug resistance

Abstract The aim of this study was to investigate the role of Notch signaling in intrinsic and bone marrow stromal cells (BMSC)-mediated drug resistance in multiple myeloma (MM) and in MM-stem cell (SC) niche maintenance. MM is an incurable hematological malignancy due to intrinsic or BMSC-mediated drug resistance; the hyperexpression of two Notch ligands, Jag1 and 2 in MM increases Notch signaling in MM cells and BMSCs resulting in malignant cells survival and proliferation. Notch pathway supports stem cell maintenance and drug resistance is an intrinsic feature of cancer stem cells; MM stem cells (MM-SCs) have been characterized as CD138- subpopulation. MM-SCs are resistant to common drugs used in therapy and responsible for disease relapse. MM cell lines were cultured alone or co-cultured with NIH3T3 murine fibroblasts or HS5 human BMSC line. To detect apoptosis induced by Mitoxantrone, Bortezomib and Melphalan, AnnexinV+ cells were processed by flow cytometry (FC). Jag1 and Jag2 were transiently silenced in MM cells using specific siRNAs. The gene expression levels were analyzed by quantitative RT-PCR. Anti-apoptotic proteins were assessed by FC. Notch inhibition was obtained by γ-secretase inhibitor and the effect on MM cell stemness potential of was assessed by FC measure of CD138- MM cells or clonogenic serial replating in methylcellulose-based medium. Our results demonstrate that Jag1 and 2 silencing reduces anti-apoptotic genes expression, i.e. SDF1α, CXCR4, Bcl-XL, Bcl2, Survivin and ABCC1 and increases sensitivity of MM cells to the used drugs. MM cells and BMSCs reciprocally activate Notch signaling resulting in increased drug resistance due to: i) an elevated expression of the anti-apoptotic genes in MM cells; ii) BMSCs release of soluble factors, i.e. SDF1α and VEGF, relevant for MM cell growth and survival. Interestingly, Jag1 and 2 silencing in MM cells co-cultured with BMSCs could reverse all gene and protein expression changes as well as BMSCs protective effect increasing the apoptotic rate of MM cells. In addition, we show in MM cell lines that DAPT-mediated Notch inhibition decreases MM-SCs and reduces the clonogenic ability in serial replating. The evidence that Jag1 and 2 silencing affects the intrinsic and BMSC-induced drug resistance in MM cells also by affecting the MM-SC population supports the rationale for a Notch-tailored approach to overcome the unavoidable relapse pf MM patient. Citation Format: Silvia Garavelli, Elisa Lazzari, Michela Colombo, Natalia Platonova, Maria Teresa Palano, Francesco Baccianti, Serena Galletti, Antonino Neri, Leslie Crews, Catriona Jamieson, Raffaella Chiaramonte. The role of NOTCH pathway in multiple myeloma associated drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-025. doi:10.1158/1538-7445.AM2017-LB-025

Abstract 4670: Identification of a possible therapeutic candidate for multiple myeloma based on dual inhibition of EZH1/EZH2

Abstract Multiple myeloma (MM) is largely incurable as the disease eventually relapses despite the recent development of novel therapies. Previous reports show that side population (SP) cells comprise myeloma stem cells. Therefore, targeting SP cells may be a promising strategy for preventing and treating MM relapse. Polycomb repressive complexes 1 (PRC1) and 2 (PRC2) are important epigenetic regulators that maintain the “stemness” of ES cells and other hematopoietic stem cells. Enhancer of zeste homolog 1 and 2 (EZH1/2) are catalytic components of PRC2, which trimethylate histone H3 at lysine 27 to repress transcription of target genes. Mutation and overexpression of EZH2 are associated with many cancers, including MM. Here, we found that SP cells expressed significantly higher levels of EZH1/2 than non-SP cells. These results suggest that overexpression of EZH1/2 is important for maintaining the stemness of MM cells and that EZH1/2 could be a potential therapeutic target. We developed a novel EZH1/2 dual inhibitor, OR-S1, and used it to investigate the effect of pharmacologic inhibition of EZH1/2 on MM. OR-S1 suppressed the proliferation of almost all MM cell lines tested, with an IC50 significantly lower than that of the specific EZH2 inhibitor, GSK126. Furthermore, flow cytometry analysis revealed that OR-S1 significantly depleted the SP cell population. RNA-seq analysis revealed that the transcriptional profiles of MM cell lines treated with OR-S1 were characterized by up-regulation of genes related to the Wnt pathway. qRT-PCR confirmed that expression of Wnt, Frizzled, and Protein kinase C family members increased markedly after exposure to OR-S1. Previous studies show that increased activation of the canonical Wnt signaling pathway down-regulates HSC self-renewal and differentiation. Therefore, we generated β-catenin-overexpressing MM cells to examine the effects of increased Wnt signaling on MM cells. Surprisingly, proliferation of these cells was significantly lower than that of control cells. These results suggested that PRC2 directly targets Wnt signaling, and that over-activation induced by EZH1/2 dual inhibition was responsible for the reduced proliferation of MM cells. Oral administration of OR-S1 to mice bearing MM xenografts led to significant impairment of subcutaneous tumors. Interestingly, long-term administration of the drug at lower doses to mice bearing orthotopic xenografts resulted in complete eradication of minimal residual disease from the bone marrow and complete cure of MM without any serious side effects. Furthermore, OR-S1 treatment of an orthotopic PDX model derived from a relapsed and heavily pretreated MM patient led to a reduction in the levels of human immunoglobulins in the serum. Taken together, these results strongly suggest that dual inhibition of EZH1/2 is a promising therapeutic approach to eradicating myeloma stem cells and could lead to important advances in the treatment of MM. Citation Format: Makoto Nakagawa, Shuhei Fujita, Daisuke Honma, Kazushi Araki, Issay Kitabayashi. Identification of a possible therapeutic candidate for multiple myeloma based on dual inhibition of EZH1/EZH2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4670. doi:10.1158/1538-7445.AM2017-4670

BTK suppresses myeloma cellular senescence through activating AKT/P27/Rb signaling.

We previously explored the role of BTK in maintaining multiple myeloma stem cells (MMSCs) self-renewal and drug-resistance. Here we investigated the elevation of BTK suppressing MM cellular senescence, a state of irreversible cellular growth arrest. We firstly discovered that an increased expression of BTK in MM samples compared to normal controls by immunohistochemistry (IHC), and significant chromosomal gain in primary samples. In addition, BTK high-expressing MM patients are associated with poor outcome in both Total Therapy 2 (TT2) and TT3 cohorts. Knockdown BTK expression by shRNA induced MM cellular senescence using β-galactosidase (SA-b-gal) staining, cell growth arrest by cell cycle staining and decreased clonogenicity while forcing BTK expression in MM cells abrogated these characteristics. We also validated this feature in mouse embryonic fibroblast cells (MEFs), which showed that elevated BTK expression was resistant to MEF senescence after serial cultivation in vitro. Further mechanism study revealed that BTK activated AKT signaling leading to down-regulation of P27 expression and hindered RB activity while AKT inhibitor, LY294002, overcame BTK-overexpression induced cellular senescence resistance. Eventually we demonstrated that BTK inhibitor, CGI-1746, induced MM cellular senescence, colony reduction and tumorigenecity inhibition in vivo. Summarily, we designate a novel mechanism of BTK in mediating MM growth, and BTK inhibitor is of great potential in vivo and in vitro suggesting BTK is a promising therapeutic target for MM.

CARs in the Lead Against Multiple Myeloma.

The recent clinical success of CD19-directed chimeric antigen receptor (CAR) T cell therapy in chronic and acute leukemia has led to increased interest in broadening this technology to other hematological malignancies and solid tumors. Now, advances are being made using CAR T cell technology to target myeloma antigens such as B cell maturation antigen (BCMA), CD138, and kappa-light chain as well as CD19 on putative myeloma stem cells. To date, only a limited number of multiple myeloma patients have received CAR T cell therapy but preliminary results have been encouraging. In this review, we summarize the recently reported results of clinical trials conducted utilizing CAR T cell therapy in multiple myeloma (MM).

Changes in Cytokine Production and Metabolism in Bone Marrow Mesenchymal Stem Cells in MGUS and Multiple Myeloma are Driven by Hypoxia Induced PADI2 Expression Altering the Transcriptome

Changes in Cytokine Production and Metabolism in Bone Marrow Mesenchymal Stem Cells in MGUS and Multiple Myeloma are Driven by Hypoxia Induced PADI2 Expression Altering the Transcriptome

Chimeric Antigen Receptors/Genetically Modified T-Cells

Multiple myeloma (MM) is a usually incurable malignancy of plasma cells. While the therapy of MM has improved greatly in the past 15 years, therapies with novel mechanisms of action are needed for MM. Allogeneic stem cell transplantation has been shown to have a potent anti-myeloma effect, and allogeneic donor lymphocyte infusions can cause remissions of MM. These results from allogeneic transplantation show that MM can be vulnerable to cellular immunotherapies, but allogeneic transplants have substantial rates of mortality and morbidity. Anti-CD19 CAR T cells have been shown to have powerful activity against B-cell malignancies. The success of anti-CD19 CAR T cells against B-cell malignancies has motivated investigators to develop genetically-modified T-cell therapies for MM. CD19 has been targeted as a therapy for multiple myeloma. A clinical trial of anti-CD19 CAR T cells for MM is underway. Part of the rationale for targeting CD19 is that CD19 might be expressed on a myeloma stem cell, which might be a mature B cell. The NY-ESO antigen has been targeted by human-leukocyte-antigen-restricted T cells in a clinical trial enrolling MM patients. B-cell maturation antigen (BCMA) is expressed by most cases of MM. We conducted the first-in-humans clinical trial of CAR T cells targeting BCMA at the National Cancer Institute. T cells expressing the CAR used in this work (CAR-BCMA) specifically recognized BCMA-expressing cells. Twelve patients received CAR-BCMA T cells on this dose-escalation trial. Among the 6 patients treated on the lowest two dose levels, limited anti-myeloma activity and mild toxicity occurred. On the third dose level, one patient obtained a very good partial remission. Two patients were treated on the fourth dose level of 9x106 CAR+T cells/kg bodyweight. Before treatment, the first patient on the fourth dose level had chemotherapy-resistant MM making up 90% of bone marrow cells. After treatment, bone marrow plasma cells became undetectable by flow cytometry, and the patient's MM entered a stringent complete remission that lasted for 17 weeks before relapse. The second patient on the fourth dose level had chemotherapy-resistant MM with 80% bone marrow plasma cells before treatment. Twenty-eight weeks after this patient received CAR-BCMA T-cells, bone marrow plasma cells were undetectable by flow cytometry, and the serum monoclonal protein had decreased by >95%. Both patients treated on the fourth dose level had toxicity consistent with cytokine-release syndrome including fever, hypotension, and dyspnea. Both patients also had prolonged cytopenias. In summary, our findings demonstrated strong anti-myeloma activity of CAR-BCMA T cells. One of the best attributes of the CAR T-cell field is that there are multiple avenues for improving CAR T-cell therapies. New CAR designs are being tested. Any part of the CAR might be improved including development of new fully-human single chain variable fragments (scFv) for the antigen-recognition component of the CAR, testing different hinge and transmembrane domains, and defining the optimal costimulatory moieties. Another avenue for improving CAR T-cell therapies is improving T-cell culture methods. Optimizing clinical application of CAR T cells, especially enhancing toxicity management, is another important avenue of improving CAR T-cell therapies. Finally, identifying new CAR target antigens is a critically important area of CAR research. In summary, genetically-modified T cells hold great promise to make a profound improvement in the therapy of multiple myeloma. Disclosures Kochenderfer: bluebird bio: Patents & Royalties, Research Funding; Kite Pharma: Patents & Royalties, Research Funding.

The myeloma stem cell concept, revisited: from phenomenology to operational terms.

The concept of the myeloma stem cell may have important therapeutic implications, yet its demonstration has been hampered by a lack of consistency in terms and definitions. Here, we summarize the current documentation and propose single-cell in vitro studies for future translational studies. By the classical approach, a CD19-/CD45low/-/CD38high/CD138+ malignant plasma cell, but not the CD19+/CD38low/- memory B cell compartment, is enriched for tumorigenic cells that initiate myeloma in xenografted immunodeficient mice, supporting that myeloma stem cells are present in the malignant PC compartment. Using a new approach, analysis of c-DNA libraries from CD19+/CD27+/CD38- single cells has identified clonotypic memory B cell, suggested to be the cell of origin. This is consistent with multiple myeloma being a multistep hierarchical process before or during clinical presentation. We anticipate that further characterization will require single cell geno- and phenotyping combined with clonogenic assays. To implement such technologies, we propose a revision of the concept of a myeloma stem cell by including operational in vitro assays to describe the cellular components of origin, initiation, maintenance, and evolution of multiple myeloma. These terms are in accordance with recent (2012) consensus statements on the definitions, assays, and nomenclature of cancer stem cells, which is technically precise without completely abolishing established terminology. We expect that this operational model will be useful for future reporting of parameters used to identify and characterize the multiple myeloma stem cells. We strongly recommend that these parameters include validated standard technologies, reproducible assays, and, most importantly, supervised prospective sampling of selected biomaterial which reflects clinical stages, disease spectrum, and therapeutic outcome. This framework is key to the characterization of the cellular architecture of multiple myeloma and its use in precision medicine.

Anti-myeloma activity of MELK inhibitor OTS167: effects on drug-resistant myeloma cells and putative myeloma stem cell replenishment of malignant plasma cells.

Currently considered incurable, multiple myeloma (MM) is characterized by proliferation of malignant plasma cells (PC) predominantly in the bone marrow, which overproduce monoclonal immunoglobulin proteins, and a perturbed tumor microenvironment, which promotes PC survival, inhibits osteoblast activity, increases osteoclast activity, and leads to hallmark osteolytic bone disease, all of which contribute to the clinical manifestations of the disease. Disrupting these sequelae by incorporation of proteasome inhibitors and immunomodulatory drugs into treatment regimens has improved overall survival of MM patients; however, the majority of patients will become refractory to the most effective currently available therapeutic options and relapse.1 It is hypothesized that a drug-resistant population of myeloma stem cells promotes disease relapse, but the characteristics and identity of this cell type(s) remain uncertain.2 Expression of maternal embryonic leucine zipper kinase (MELK) is increased in a number of cancers and is associated with poorer prognosis. MELK activity modulates many cellular and biological processes, including proliferation, apoptosis, hematopoiesis and oncogenesis, and is believed to have a critical role in cancer stem cell maintenance.3

Citrullination of histone H3 drives IL-6 production by bone marrow mesenchymal stem cells in MGUS and multiple myeloma

Multiple myeloma (MM), an incurable plasma cell malignancy, requires localisation within the bone marrow. This microenvironment facilitates crucial interactions between the cancer cells and stromal cell types that permit the tumour to survive and proliferate. There is increasing evidence that the bone marrow mesenchymal stem cell (BMMSC) is stably altered in patients with MM—a phenotype also postulated to exist in patients with monoclonal gammopathy of undetermined significance (MGUS) a benign condition that precedes MM. In this study, we describe a mechanism by which increased expression of peptidyl arginine deiminase 2 (PADI2) by BMMSCs in patients with MGUS and MM directly alters malignant plasma cell phenotype. We identify PADI2 as one of the most highly upregulated transcripts in BMMSCs from both MGUS and MM patients, and that through its enzymatic deimination of histone H3 arginine 26, PADI2 activity directly induces the upregulation of interleukin-6 expression. This leads to the acquisition of resistance to the chemotherapeutic agent, bortezomib, by malignant plasma cells. We therefore describe a novel mechanism by which BMMSC dysfunction in patients with MGUS and MM directly leads to pro-malignancy signalling through the citrullination of histone H3R26.

Low-Dose Cyclophosphamide Plus Granulocyte Colony-Stimulating Factor As an Efficacious and Safe Peripheral Blood Stem Cell Mobilizing Regimen in Patients with Hematologic Malignancies

▪Introduction:Collection of adequate numbers of Hematopoietic Stem Cells (HSCs) is a prerequisite for proceeding to autologous bone marrow transplant. However, various studies have showed that approximately 5% to 40% of patients do not meet the minimum threshold of 2×106 CD34+ cells/kg that is associated with timely engraftment and better outcomes. There is little consensus over the optimal mobilization regimen for procurement of peripheral blood CD34 + stem cells (PBSCs). Studies comparing mobilization using high-dose cyclophosphamide(HD-CY) {5-7 g/m2} or intermediate dose cyclophosphamide (ID-CY) {3-4g/m2} plus G-CSF with low dose cyclophosphamide (LD-CY) {1-2g/m2) plus G-CSF have reported higher total PBSC yield with the former but at the cost of higher toxicity [Hiwase et al, Cytotherapy 2007]. Hence the significance of our study to evaluate the efficacy and safety of LD-CY plus G-CSF in the era of novel induction therapies, takes precedence in terms of optimal resource utilization.Methods:We retrospectively analyzed mobilization efficacy and need for supportive care in Myeloma and Lymphoma patients that received LD-CY (2g/m2) plus G-CSF (10µg/kg/day) as the preferred mobilization regimen. Patients treated with novel induction regimens only, with or without radiation were included. LD-CY was given on day 1 with MESNA. G-CSF was started Day +5 from LD-CY and continued until the completion of apheresis. We started to measure peripheral CD34+ (pCD34) when patient's white blood cell count recovered to >1000/µL. When the pCD34 count was ≥10/uL, apheresis was started.Results:Out of 21 patients analyzed, 14 (67%) were Myeloma, predominantly IgG subtype (71%) and 7(33%) Lymphoma predominantly Non-Hodgkins type (71%). Our study population comprised of 53% females, 81% Caucasians with median age of 59 years (30-66). 28.5% patients had radiation treatment in the past. Mean/Median lines of chemotherapy previously received were 2/1 (1-5).71% patients received Lenalidomide (57%) / Thalidomide (14%) based regimens for induction therapies. 52% patients had complete response at the time of chemo mobilization.Successful mobilization (defined as total CD34+ cells collected >2x106/kg) was significantly achieved in 95% patients with mean/ median collection of 12.4/11.5 x106 CD34+ cells/kg (20 out of 21 patients, One sample T- test with significance level, alpha =0.025 [one sided ]showed T statistic 5.36 with p-value <0.0001). First apheresis yielded at least 2 x10 6/kg CD34+ cells in 76 % patients with a mean/median of 5.8/3.8 x106/kg CD34+ cells. 66% of patients were found to be good mobilizers, defined as patients collecting ≥5 x106 CD34+ cells/kg in ≤2 days. 81% patients collected ≥5x106 CD34+ cells/kg and 57 % patients collected ≥10 x106 CD34+ cells/kg. Mean/Median Peak pCD34count was 97.7/60 cells/µL(2-488). Median number of aphereses was 2 (mean 2.6) with the mean and median time to apheresis being 11 days (9-15). None of the patients needed inpatient hospitalization or intravenous antibiotics during mobilization or had any complication related to immunosuppression. 14% and 19% of patients needed packed red blood cell and platelet transfusions respectively during the mobilization period. One Myeloma patient (4.8%) that failed mobilization had previously received five lines of chemotherapy including lenalidomide/thalidomide containing regimens and had stable disease at the time of mobilization. Interestingly, 61% of myeloma patients that successfully mobilized had received lenalidomide containing induction regimen (mean/median no. of cycles- 3.9/4[1-7])Conclusion:Our analysis shows that LD-CY plus G-CSF is efficacious to mobilize sufficient number of stem cells (>2x106 CD34+ cells /kg) in Myeloma and Lymphoma patients treated with novel agents, including the challenging sub-group of lenalidomide treated myeloma patients, with no infectious complications or morbidity noted during the entire period of mobilization. Therefore, we believe LD-CY plus G-CSF may overcome the negative effects of prior lenalidomide exposure on PBSC mobilization [Mark et al, Biol Blood Marrow Transplant 2008] and is superior in terms of optimal resource utilization compared to novel plerixafor based regimens [Chaudhary et al, J Clin Apher 2013]. Prospective studies involving CY based regimens will help elucidate the optimal dose of chemo-mobilization. DisclosuresNo relevant conflicts of interest to declare.

Phospho-Flow Cytometry Quantitation of STAT Signalling Checkpoints in Malignant and Non-Malignant Cells Identifies Patients Suitable for pSTAT3 and 5 Targeted Inhibitors

BackgroundIL-6 receptor/JAK/STAT is a major signalling pathway associated with pleiotropic functions including cell transformation and proliferation. STAT3, one of a family of 7 STAT members, sits at the apex of the signalling cascade and is a potential target for cancer therapy. Small molecular weight inhibitors either prevent STAT3 phosphorylation or target high constitutive levels of phosphorylated pSTAT3 (pSTAT3). Increased constitutive expression of pSTAT3 is a hallmark of solid tumors and is associated with increased morbidity. However recent studies suggest that this may not apply to hematological malignancies. Phospho-flow cytometry offers a novel approach for the quantitation of constitutive and cytokine induced pSTAT3 expression in subsets of hemopoietic cells. Constitutive pSTAT3 expression is not a prognostic factor for patients with either multiple myeloma (MM) or acute myeloid leukemia (AML), though we recently demonstrated that IL-6 induced pSTAT3 was associated with better prognosis in MM (χ2 =13.06; p<0.0003) (Leukemia 2015; 29:483) . There is considerable heterogeneity of pSTAT3 expression between patients which suggests that quantitation of these new biomarkers may help to identify those patients most likely to respond to STAT-targeted therapy. MethodsWe have determined constitutive and IL-6 induced pSTAT3 and 5 in malignant plasma cells of patients with MM (n=25) and for comparison, the blast cells in AML (n=8) and CD5+ B cells in chronic lymphocytic leukemia (CLL) (n=12) as well as the non-malignant B and T cells in each sample. Normal BM cells (n=9) served as a control. We have also determined pSTAT3 and 5 expression in "side population" cells (SP), the putative myeloma stem cells, and determined whether MM and AML tumor cells induce pSTAT3 on normal lymphocytes in vitro by co-culture experiments. IL-6 receptor expression was also determined on all cells.ResultsConstitutive pSTAT3 expression was increased (>5% of normal lymphocytes) in the malignant plasma cells of 44% of patients with MM but by comparison was not increased in AML or B-CLL. Constitutive pSTAT5 was increased (>5%) in 68% of MM plasma cells and 75% of AML cells and was either normal or decreased in CLL cells. Patients with increased pSTAT3 or 5 in MM cells also had high expression in autologous normal T cells. The level of constitutive pSTAT3 and 5 was not related to the disease status (relapse vs remission). In patients with MM, IL-6 induced an increased pSTAT3 expression (induced/constitutive ratio >1.5) in both MM cells and T cells in 88% of samples. IL-6 induced pSTAT3 expression in blasts of 89% of AML patients (median ratio = 79). There was no correlation between the level of pSTAT3 induced by IL-6 and IL-6 receptor expression. IL-6 did not increase pSTAT5 levels in MM, AML or CLL. SP cells in the MM cell line RPMI 8226 had lower constitutive pSTAT3 than the non-SP population and a lower IL-6 receptor expression, but had a greater response to cytokine stimulation, suggesting that SP cells may be more sensitive than non-SP cells to inhibitors of STAT phosphorylation. Addition of RPMI 8226 cells or supernatant to normal T cells caused an increase in pSTAT3, suggesting that the upregulation of pSTAT3 is at least partly due to a tumor-derived soluble agent in the microenvironment.ConclusionsThese results demonstrate the heterogeneity of constitutive and cytokine induced pSTAT3 and 5 expression in MM, AML and CLL. Elevated levels of constitutive pSTAT3 (approx 44% of MM) and pSTAT5 (88% of MM and AML) in the malignant clone were associated with high levels in autologous non-malignant T cells and are not of prognostic significance. Therefore elevated pSTAT3 and 5 may not be an exclusive hallmark of the malignant clone, as reported in solid tumors, but rather a result of the microenvironment and a high IL6-R expression. Phospho-flow cytometry enables precise quantitation of constitutive and cytokine-induced pSTAT3 and 5, and thus can identify the patients most likely to respond to direct STAT-3 or 5 inhibitor therapies. SP cells (putative MM stem cells) appear to be potentially targetable. Inhibitors targeting signalling pathways may be more efficacious if therapy is directed to patients with targetable checkpoints. DisclosuresHart:DendroCyte BioTech Pty Ltd: Equity Ownership.

Immunotherapy in Multiple Myeloma Using Cancer-Testis Antigens.

Context:Multiple myeloma (MM) is a B-cell malignancy characterized by monoclonal expansion of abnormal plasma cells in the bone marrow. It accounts for 10% of hematological malignancies. Although patients respond to a wide range of anticancer modalities, relapse occurs in a significant number of the cases. Immunotherapeutic approaches have been evolved to tackle this problem. Cancer-testis antigens CTAs as a group of tumor-associated antigens are appropriate targets for cancer immunotherapy as they have restricted expression pattern in normal tissues except for testis which is an immune-privileged site. Expression of these antigens has been assessed in different malignancies including MM.Evidence Acquisition:We performed a computerized search of the MEDLINE/PubMed databases with key words: multiple myeloma, cancer-testis antigen, and cancer stem cell and immunotherapy.Results:Several CTAs including NY-ESO-1, MAGE and GAGE family have been shown to be expressed in MM patients. Cellular and humoral immune responses against these antigens have been detected in MM patients.Conclusions:The frequent and high expression level of CTAs in MM patients shows that these antigens can be applied as cancer biomarkers as well as targets for immunotherapy in these patients.

Cancer stem cells are the cause of drug resistance in multiple myeloma: fact or fiction?

Multiple myeloma (MM) remains a largely incurable, genetically heterogeneous plasma-cell malignancy that contains - just like many other cancers - a small fraction of clonogenic stem cell-like cells that exhibit pronounced self-renewal and differentiation capacities, but also pronounced drug resistance. These MM stem cells (MMSCs) are a controversial but highly significant issue in myeloma research because, in our opinion, they are at the root of the failure of anti-neoplastic chemotherapies to transform myeloma to a manageable chronic disease. Several markers including CD138-, ALDH1+ and SP have been used to identify MMSCs; however, no single marker is reliable for the isolation of MMSC. Nonetheless, it is now known that MMSCs depend on self-renewal and pro-survival pathways, such as AKT, Wnt/β-catenin, Notch and Hedgehog, which can be targeted with novel drugs that have shown promise in pre-clinical and clinical trials. Here, we review the pathways of myeloma "stemness", the interactions with the bone marrow microenvironment that promote drug resistance, and the obstacles that must be overcome to eradicate MMSCs and make myeloma a curable disease.

Multiple myeloma and bone marrow mesenchymal stem cells' crosstalk: Effect on translation initiation.

Multiple myeloma (MM) malignant plasma cells reside in the bone marrow (BM) and convert it into a specialized pre-neoplastic niche that promotes the proliferation and survival of the cancer cells. BM resident mesenchymal stem cells (BM-MSCs) are altered in MM and in vitro studies indicate their transformation by MM proximity is within hours. The response time frame suggested that protein translation may be implicated. Thus, we assembled a co-culture model of MM cell lines with MSCs from normal donors (ND) and MM patients to test our hypothesis. The cell lines (U266, ARP-1) and BM-MSCs (ND, MM) were harvested separately after 72 h of co-culture and assayed for proliferation, death, levels of major translation initiation factors (eIF4E, eIF4GI), their targets, and regulators. Significant changes were observed: BM-MSCs (ND and MM) co-cultured with MM cell lines displayed elevated proliferation and death as well as increased expression/activity of eIF4E/eIF4GI; MM cell lines co-cultured with MM-MSCs also displayed higher proliferation and death rates coupled with augmented translation initiation factors; in contrast, MM cell lines co-cultured with ND-MSCs did not display elevated proliferation only death and had no changes in eIF4GI levels/activity. eIF4E expression was increased in one of the cell lines. Our study demonstrates that there is direct dialogue between the MM and BM-MSCs populations that includes translation initiation manipulation and critically affects cell fate. Future research should be aimed at identifying therapeutic targets that may be used to minimize the collateral damage to the cancer microenvironment and limit its recruitment into the malignant process. © 2015 Wiley Periodicals, Inc.

MicroRNA-451 regulates stemness of side population cells via PI3K/Akt/mTOR signaling pathway in multiple myeloma.

Side population (SP) cells are an enriched source of cancer-initiating cells with stemness characteristics, generated by increased ABC transporter activity, which has served as a unique hallmark for multiple myeloma (MM) stem cell studies. Here we isolated and identified MM SP cells via Hoechst 33342 staining. Furthermore, we demonstrate that SP cells possess abnormal cell cycle, clonogenicity, and high drug efflux characteristics-all of which are features commonly seen in stem cells. Interestingly, we found that bortezomib, As2O3, and melphalan all affected apoptosis and clonogenicity in SP cells. We followed by characterizing the miRNA signature of MM SP cells and validated the specific miR-451 target tuberous sclerosis 1 (TSC1) gene to reveal that it activates the PI3K/Akt/mTOR signaling in MM SP cells. Inhibition of miR-451 enhanced anti-myeloma novel agents' effectiveness, through increasing cells apoptosis, decreasing clonogenicity, and reducing MDR1 mRNA expression. Moreover, the novel specific PI3K/Akt/mTOR signaling inhibitor S14161 displayed its prowess as a potential therapeutic agent by targeting MM SP cells. Our findings offer insights into the mechanisms regulating MM SP cells and provide a novel strategy to overcome resistance to existing therapies against myeloma.

Whole-epigenome analysis in multiple myeloma reveals DNA hypermethylation of B cell-specific enhancers.

While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with down-regulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cell-specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM.

Bruton tyrosine kinase is a therapeutic target in stem-like cells from multiple myeloma.

Ibrutinib (Imbruvica), a small-drug inhibitor of Bruton tyrosine kinase (BTK), is currently undergoing clinical testing in patients with multiple myeloma, yet important questions on the role of BTK in myeloma biology and treatment are outstanding. Using flow-sorted side population cells from human myeloma cell lines and multiple myeloma primary samples as surrogate for the elusive multiple myeloma stem cell, we found that elevated expression of BTK in myeloma cells leads to AKT/WNT/β-catenin-dependent upregulation of key stemness genes (OCT4, SOX2, NANOG, and MYC) and enhanced self-renewal. Enforced transgenic expression of BTK in myeloma cells increased features of cancer stemness, including clonogenicity and resistance to widely used myeloma drugs, whereas inducible knockdown of BTK abolished them. Furthermore, overexpression of BTK in myeloma cells promoted tumor growth in laboratory mice and rendered side population-derived tumors that contained high levels of BTK more sensitive to the selective, second-generation BTK inhibitor, CGI1746, than side population-derived tumors that harbored low levels of BTK. Taken together, these findings implicate BTK as a positive regulator of myeloma stemness and provide additional support for the clinical testing of BTK-targeted therapies in patients with myeloma.

Mobilization and transplantation patterns of autologous hematopoietic stem cells in multiple myeloma and non-Hodgkin lymphoma.

The mobilization of hematopoietic stem cells can be a limiting factor for transplantation, yet little is known about how the availability of novel mobilizing agents has affected the practices of oncologists and transplant specialists. US-based oncologists (n = 48) and transplant specialists (n = 46) were separately surveyed with a partial overlap of assessed information. More transplant specialists than oncologists believed that the time between referral and first consultation is adequate (89.1% vs 54.2%; P < .001). The presence of comorbidities was the most common reason for patients not being referred for transplantation. Among oncologists, 31.3% avoided cyclophosphamide and 16.7% avoided lenalidomide to prevent mobilization impairment in patients with multiple myeloma (MM). Chemotherapy mobilization for MM was used by 23.9% of transplant specialists due to higher CD34+ yields and by 21.7% due to its anti-MM effect. In non-Hodgkin lymphoma (NHL), 26.1% of transplant specialists used chemotherapy mobilization due to higher CD34+ yields, and 26.1% collected hematopoietic stem cells on the rebound prior to chemotherapy. With regard to plerixafor use in MM, 36.9% of transplant specialists reported that they did not use it, and 28.3% said they reserved it for second mobilization. In NHL, 4.3% of transplant specialists reported not using plerixafor, and 39.1% reserved it for second mobilization. Educational needs were identified to promote adequate referral for transplantation as well as successful and cost-effective methods for the mobilization of hematopoietic stem cells.

Side Population of Multiple Myeloma and Multiple Myeloma Stem Cell

BackgroundSide population (SP) cells are known as enriched source of cancer initiating cells with stem cell properties. The SP cells show a distinct low-staining pattern with the Hoechst 33342 dye. Our aim was to identify SP cells in multiple myeloma (MM) cell lines as well as characteristics of SP cells. Furthermore, we investigated drugs that are effective against SP cells of MM.MethodsSP (side population), MP (main population) cells from human multiple myeloma cell line (RPMI8226) were sorted by flow cytometry based Hoechst 33342 staining. We performed proliferative capacity in vitro, colony-forming cell assay, cell cycle analysis, drug toxicity (doxorubicin, dexamethasone, bortezomib, melphalan). Expression of stem cell markers (ABCG2, Oct4, Sox2, and Nanog) was observed by western blotting assay.ResultsSeveral established studies have shown a clonogenic MM cell population with CD 138-status. Therefore, we assessed whether there is correlation between CD 138 - subpopulations and SP cells. CD 138- subpopulations of MM cell lines were not consistent with SP cells. And we have found no correlation between expression of CD 19, CD20, and CD 27 and the proportion of SP cells. No difference was observed for expression of stem cell marker between the SP cells and MP cells. We examined cell cycle analysis and proliferation ability of SP and MP cells. SP cells showed a capacity for higher proliferation abilities and clonogenecity than MP cells. (Fig1,2)We investigated target SP cells with several drugs (doxorubicin, dexamethasone, melphalan, bortezomib). Importantly, bortezomib was the most effective drug for SP cells.(Table1)ConclusionIn our study, there was lack of evidence that SP cells was consistent with MM stem cells. No difference was observed for any other marker between SP cells and MP cells. However SP cells had higher proliferation index and clonogenicity compared with MP cells. We suggest that SP cells are rather related to resistance of drugs. Furthermore, bortezomib reduced the SP cells more effectively compared to other drugs. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Impact of Primary Prophylaxis with Granulocyte Colony-Stimulating Factors on Febrile Neutropenia during Chemotherapy: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

Introduction Febrile neutropenia (FN) is a dose-limiting toxicity of myelosuppressive chemotherapy that has been associated with decreased chemotherapy relative dose intensity (RDI) and increased morbidity and mortality. In clinical trials, primary prophylaxis (PP) with recombinant human granulocyte colony-stimulating factors (G-CSFs) has been shown to reduce the risk of FN, chemotherapy dose delays/reductions, decreased RDI, antibiotic use, and FN-related hospitalization. This systematic review and meta-analysis assessed the relative efficacy of PP with different G-CSFs to reduce the incidence of FN in cancer patients who received myelosuppressive chemotherapy in randomized controlled trials (RCTs).Methods A systematic literature review identified publications (January 1990 to September 2013) of RCTs assessing PP with filgrastim, pegfilgrastim, lenograstim, or lipegfilgrastim versus placebo, no G-CSF PP, or a different G-CSF in adults who received myelosuppressive chemotherapy for solid tumors or non-Hodgkin’s lymphoma. Terms for G-CSFs were searched for in PubMed, Embase, Science Citation Index, Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials, Database of Abstracts of Reviews of Effects, Health Technology Assessment Database, and the National Health Service Economic Evaluation Database; original publications were searched manually. Eligible studies enrolled patients who initiated G-CSF PP 1–3 days after completing chemotherapy in each cycle; control patients were eligible for secondary prophylaxis with the same G-CSF after the first cycle. Study exclusion criteria included granulocyte-macrophage colony-stimulating factor use, leukemia or multiple myeloma, bone marrow or peripheral blood stem cell transplantation, G-CSF for established FN, different doses of the same G-CSF in each treatment arm, and investigational or unapproved drugs. Economic analyses and studies published in languages other than English were excluded. A meta-analysis using mixed-treatment comparison (MTC) was used to calculate the odds ratio (OR) and 95% credible interval of FN in all chemotherapy cycles and in cycle 1 without adjustment for differences in RDI between study treatment arms. No adjustment for differences in RDI between arms was made because RDI was not consistently reported between studies.Results Of the 4790 publications initially screened, 27 publications representing 30 studies were included in the meta-analysis (Figure). Over all chemotherapy cycles, there was a statistically significant reduction in the FN risk for PP with filgrastim, pegfilgrastim, lenograstim, and lipegfilgrastim versus no G-CSF PP or placebo, as well as with pegfilgrastim PP versus filgrastim PP (Table). Over all chemotherapy cycles, there was a statistically nonsignificant increase in the FN risk for lipegfilgrastim PP versus pegfilgrastim PP; a statistically significant difference was not expected because of the small sample size (n=306) for lipegfilgrastim (2 studies). During chemotherapy cycle 1, there was a statistically significant reduction in the FN risk for filgrastim PP versus no G-CSF PP or placebo, pegfilgrastim PP versus no G-CSF PP or placebo, and lipegfilgrastim PP versus no G-CSF or placebo (data not shown).Conclusions Using MTC without adjustment for RDI, PP with short-acting and long-acting G-CSFs was associated with a reduced FN risk in patients receiving myelosuppressive chemotherapy for solid tumors or non-Hodgkin’s lymphoma. In future studies, consistent reporting of RDI between study arms is needed to adequately assess the influence of RDI on FN outcomes and to eliminate the potential bias in comparisons between G-CSF arms receiving more intensive chemotherapy than control arms. [Display omitted] TablePrimary prophylaxis comparisons across all cyclesMedian OR (95% CrI)Filgrastim vs no G-CSF or placebo (11 studies; n=2181)0.42 (0.30–0.57)Pegfilgrastim vs no G-CSF or placebo (5 studies; n=2060)0.25 (0.17–0.40)Lenograstim vs no G-CSF or placebo (5 studies; n=467)0.34 (0.19–0.60)Lipegfilgrastim vs no G-CSF or placebo (1 study; n=375)0.35 (0.14–0.88)Pegfilgrastim vs filgrastim (6 studies; n=647)0.61 (0.40–0.98)Lipegfilgrastim vs pegfilgrastim (2 studies; n=306)1.39 (0.54–3.50)Heterogeneity, between-trial SD logOR, mean (95% CI)0.41 (0.17–0.69)Mean residual difference60.01 DisclosuresWang:Amgen Inc.: Consultancy. Baser:Amgen Inc.: Consultancy. Kutikova:Amgen Inc.: Employment, Equity Ownership. Page:Amgen Inc.: Employment, Equity Ownership. Barron:Amgen Inc.: Employment, Equity Ownership.