Role of Mir-30e in Multiple Myeloma Cells Resistance to Lenalidomide and Bortezomib

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 323-323
Author(s):  
Paola Neri ◽  
Kathy Gratton ◽  
Li Ren ◽  
Jordan Johnson ◽  
Jiri Slaby ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mtt Assay ◽  
Research Funding ◽  
Plasma Cells ◽  
Response To Therapy ◽  
Transcriptional Level ◽  
Seed Region ◽  
Mrna Levels ◽  
Protein Levels ◽  
Empty Vector

Abstract Abstract 323 Background: miRNAs are non-coding small RNAs that modulate protein expression at the post-transcriptional level and are implicated in the pathogenesis of a variety of cancers. In Multiple Myeloma (MM) a global elevation of miRNAs was previously correlated with poor disease outcomes and response to therapy. Using miRNome profiling of MM patients, we have recently established a miRNA-based risk score that is predictive of response to lenalidomide (Neri P, Blood 2011). In particular, we identified significant upregulation of miR-30 family members (a, b, c and e) in lenalidomide resistant patients. In the present study, we evaluated the biological functions of miR-30e in MM and its role in plasma cells resistance to lenalidomide as well as other anti-MM therapeutics. Methods and Results: Microarray profiling (Affymetrix miRNA GeneChip) of total RNA extracted from bone marrow plasma cells from lenalidomide sensitive and resistant MM patients (n=40), coupled with quantitative short stem-loop PCR (TaqMan, Applied Biosystems), confirmed the upregulation of miR-30e in lenalidomide resistant patients. Functionally, we sought to determine if overexpression of miR-30e would modify MM cells sensitivity to lenalidomide and bortezomib. Lentiviral-mediated stable expression (pLKO.1 retroviral plasmid) of miR-30e, and relative to empty vector (EV), significant increased MM1S and OPM2 cells growth (1.3 fold) as determined by MTT assay. In addition, miR-30e overexpressing cells (MM1S-30e and OPM2-30e vs MM1-EV and OPM2-EV) were more resistant to the cytotoxic effects of lenalidomide as well as bortezomib with approximately 15 to 20% reduction in cells death (Annexin V staining and MTT assay). Computational target prediction analysis (TargetScan 6.0 and miRanda) identified CRBN and BLIMP1 as potential target of miR-30e with a miRNA seed region that matches 8 or 7mer sites within Cereblon and BLIMP1 3'UTR regions. In a panel of MM cell lines (MM1S, OPM2, H929, INA-6, U266, 8226, KMS11) CRBN mRNA levels were indeed inversely correlated with miR-30e and stable mir-30e overexpression significantly reduced CRBN mRNA in these cells (MM1S-30e and OPM2-30e). In addition to CRBN, BLIMP1 mRNA and protein levels were also reduced in miR-30e overexpressing cells. In plasma cells, BLIMP1 drives XBP1 expression while supressing c-myc. In MM1S-30e and OPM2-30e (relative to empty vector), and consistent with their reduced BLIMP1 expression, XBP1 mRNA and protein levels were reduced. Furthermore, treatment with lenalidomide (10μM) significantly reduced c-MYC protein levels in MM1S-EV cells after 4 hours while it had no effect on C-MYC expression in MM1S-30e cells. Conclusions: miR-30e is overexpressed in resistant MM cells and is here shown to regulate cereblon expression, plasma cells differentiation axis (BLIMP1, XBP1) and cell growth (c-MYC). Disclosures: Neri: Johnson ans Johnson: Research Funding. Bahlis:Johnson and Johnson: Honoraria, Research Funding; Celgene: Honoraria.

scholarly journals Gfi1 Transcription Factor Is a Pro-Survival Molecule in Multiple Myeloma Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4422-4422 ◽  
Author(s):  
Denise Toscani ◽  
Cheolkyu Park ◽  
Fengming Wang ◽  
Judith Anderson ◽  
Nicola Giuliani ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Research Funding ◽  
Caspase 3 ◽  
Mrna Levels ◽  
Gene Promoters ◽  
Protein Levels ◽  
Empty Vector ◽  
Apoptotic Effects ◽  
Mtt Assays

Abstract Rational: Multiple myeloma (MM) is the second most common hematologic malignancy and is incurable for most patients. We reported that MM cells induce expression of the transcriptional repressor, Growth independent factor 1 (Gfi1), in bone marrow stromal cells (BMSC) repressing Runx2 gene transcription, and results in prolonged suppression of osteoblast differentiation. Since Gfi1 is an anti-apoptotic factor in other hematologic malignancies, we hypothesized that Gfi1 has an important pro-survival role in MM cells by blocking apoptosis and can attenuate the pro-apoptotic effects of bortezomib. Methods: CD138+ cells isolated from MM patients, healthy donors and human MM cell lines (HMCLs) (H929, JJN3, and MM1.S) were tested for Gfi1 expression levels and the effects of Gfi1 knock down (KD) on MM cell survival by transduction with pLKO.1-puro lentivirus vectors encoding Gfi1 or non-mammalian shRNAs. HMCLs were treated with IL-6, S1P or TNFa or co-cultured with a human BMSC line (SAKA-T) to assess their effects on Gfi1 expression. The anti-apoptotic effects of Gfi1 overexpression (o/e) in MM1.S and H929 were tested by transduction with a pUC2 lentivirus encoding Gfi1 or with the empty vector followed by bortezomib (2 - 5nM) or vehicle (DMSO) treatment for 24 and 48 hours. MTT assays and cleaved caspase 3 protein levels were used to assess cell viability and cell death. Since acetylation of Gfi1 and p53 affects their activity and ability to bind each other, we also characterized HDAC inhibitors (HDACi)-induced changes on p53 enrichment at the Noxa, PUMA and p21 gene promoters by ChIP assays and the effects of acetylation of Gfi1 on its p53 binding capacity in MM cells. Results:We found that Gfi1 is highly expressed, at the mRNA and protein level, in CD138+ cells from MM patients and cell lines than CD138+ cells from normal donors. Gfi1 expression was further increased in MM cells by exogenous IL-6 (5ng/ml) and sphingosine-1-phosphate (S1P) (0.1 µM), but not by TNFα (10 ng/ml). KD of Gfi1 inhibited the growth and induced apoptosis of MM cells, as measured by increased mRNA levels of Bax, PUMA, Noxa, increased cleaved caspase 3 protein levels and decreased protein levels of Mcl-1 and c-Myc. Gfi1 (o/e) in MM cells conferred a survival advantage over their respective empty vector transduced controls as assessed by cell counts and MTT assays. Further, Gfi1 o/e protected MM cells from apoptosis induced by treatment with bortezomib as measured by MTT and cleaved caspase 3 protein levels. Since SphK1 activitycan also prevent apoptosis of MM cells, we next determined if Gfi1 regulated SphK1 in MM cells.CD138+ cells from MM patients had increased SphK1 mRNA levels compared with normal donors, and SphK1 mRNA levels and protein activity were further increased in MM cells by exogenous IL-6 and S1P. Co-culture of MM cells with BMSC also enhanced Gfi1, IL6 (3 fold) and SphK1 (2.5 fold) mRNA levels in MM cells. Importantly, Gfi1 KD in MM cells profoundly downregulated SphK1 mRNA levels and reduced expression of phospho-SphK1, suggesting that Gfi1 enhances MM growth in part via increasing expression and activity of SphK1. Gfi1's inhibition of apoptosis resulted in part from binding of Gfi1 to p53, which blocked p53's access to its pro-apoptotic target gene promoters. HDACi treatment resulted in acetylation of Gfi1 and inhibited Gfi1's suppression of apoptosis by preventing Gfi1-p53 binding and subsequent enrichment of p53 at the Noxa, PUMA and p21 promoters in MM cells. Conclusion: Taken together, our results suggest that Gfi1 may act as a key regulator of MM growth and survival through its regulation of p53 and SphK1 activity, and that targeting Gfi1 may be a novel therapeutic strategy for MM patients. Disclosures Giuliani: Celgene: Research Funding; Janssen: Research Funding. Roodman:Amgen: Consultancy.

scholarly journals Single Cell Multi-Omic Profiling of Multiple Myeloma with t(4;14) Finds an Immune Microenvironment Gene Signature That Correlates with Clinical Outcomes

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2653-2653
Author(s):  
Sanjay De Mel ◽  
Jonathan Adam Scolnick ◽  
Chern Han Yong ◽  
Xiaojing Huo ◽  
Stacy Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Protein Expression ◽  
Single Cell ◽  
Research Funding ◽  
Plasma Cells ◽  
Expression Patterns ◽  
Gene Signature ◽  
Response To Therapy ◽  
Diagnostic Samples

Abstract Background Multiple Myeloma (MM) is an incurable plasma cell (PC) malignancy and high risk MM remains an unmet clinical need. Translocation 4;14 occurs in 15% of MM and is associated with an adverse prognosis. A deeper understanding of the biology and immune micro-environment of t(4;14) MM is necessary for the development of effective targeted therapies. Single Cell multi-omics provides a new tool for phenotypic characterization of MM. Here we used Proteona's ESCAPE™ single cell multi-omics platform to study a cohort of patients with t(4;14) MM. Methods Diagnostic bone marrow (BM) samples from 13 patients with t(4;14) MM (one of whom had samples at diagnosis and relapse) were analysed using the ESCAPE™ platform from Proteona which simultaneously measures gene and cell surface protein expression of 65 proteins in single cells. Cryopreserved BM samples were stained with antibodies and subsequently sorted on CD138 expression. The CD138 positive and negative fractions were recombined at a 1:1 ratio for analysis using the 10x Genomics 3' RNAseq kit. Resulting data were analyzed with Proteona's MapSuite™ single cell analytics platform. In particular, Mapcell was used to annotate the cells and MapBatch was used for batch normalization in order to preserve rare cell populations. Results Patients had a median age of 63 years and received novel agent-based induction. Median progression free and overall survival (PFS and OS) were 22 and 34 months respectively. We first analyzed serial BM samples from an individual patient that were taken at diagnosis and relapse following bortezomib based treatment. The PCs in this patient showed variations in gene expression between diagnosis and relapse (Fig 1A), including the reduction of HIST1H2BG expression, which has previously been correlated with resistance to bortezomib. Subsequent analysis of the immune cells identified a shift in the ratio of T cells to CD14 monocytes from 5.7 at diagnosis to 0.6 at relapse suggesting a major change in the BM immune micro-environment in response to therapy. Next, we analyzed the malignant PCs of the diagnostic samples. As expected, MMSET (NSD2) was overexpressed in all PCs compared to normal PCs, while FGFR3 expression could be categorized into no expression of FGFR3, low expression (<10% of cells expressing FGFR3) or high expression (>80% of cells expressing FGFR3) (Fig 1B). No gene or protein expression patterns within the PCs were identified that correlated with PFS or OS in this cohort. Finally, we analyzed the immune micro-environment in the diagnostic samples (Fig 1C). While there was no overall discernable pattern of cell types present, one cluster of cells, annotated as 'unknown' cell type, suggested a small population of cells that had not been previously annotated in published single cell RNA-seq data. The cells were CD45+ and CD138 - both at the protein and RNA level, suggesting they are not plasma cells. We tested if the number of the 'unknown' cells in each sample correlated with PFS, but there was no significant correlation. We then used these cells to derive a gene signature profile which was expressed in most of the cells in the 'unknown' cluster as well as a minor fraction of cells in other clusters including some PCs. The number of cells expressing the gene signature negatively correlated with PFS, with samples containing more cells expressing the signature having a lower PFS than samples with fewer signature positive cells (Fig 2). The correlation remained significant whether we included PCs in the analysis or not, but was not significant amongst only the PC population, suggesting that the cells responsible for the correlation are from the immune micro-environment. Conclusions We present the first application of single cell multi-omic immune profiling in high-risk MM and demonstrate that t(4;14) is a phenotypically heterogenous disease. While no consistent gene or protein expression patterns were identified within the malignant cell population, we did identify gene expression changes in a relapsed patient sample that may reflect key alterations in the PCs responsible for therapy resistance. In addition, we identified a gene signature expressed in a rare population of non-plasma cells that significantly correlated with PFS in this patient cohort. These data highlight the potential of single cell multi-omic analysis to identify immune micro-environmental signatures that correlate with response to therapy in t(4;14) MM. Figure 1 Figure 1. Disclosures Scolnick: Proteona Pte Ltd: Current holder of individual stocks in a privately-held company. Huo: Proteona Pte Ltd: Ended employment in the past 24 months. Xu: Proteona Pte Ltd: Current Employment. Chng: Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Abbvie: Honoraria.

Tumor Suppressor MiR-29b Targets HDAC4 and Modulates Vorinostat Activity In Multiple Myeloma Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4451-4451 ◽  
Author(s):  
Nicola Amodio ◽  
Marzia Leotta ◽  
Lavinia Biamonte ◽  
Teresa Calimeri ◽  
Eugenio Morelli ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Suppressor ◽  
Conflicts Of Interest ◽  
Constitutive Expression ◽  
Microarray Dataset ◽  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Over Expression ◽  
Protein Levels

MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression at the post-transcriptional level by inducing mRNA decay or translation repression. A subclass of miRNAs, named epi-miRNAs, is known to exert anti-tumor activity by targeting effectors of the epigenetic machinery. We recently demonstrated a key role for the tumor suppressor miR-29b in reducing the global DNA methylation of multiple myeloma (MM) cells through the targeting of DNA-methyltransferases. In silico search of additional miR-29b targets contributing to clarify its role as epi-miRNA unveiled the class II histone deacetylase HDAC4. Since histone deacetylases represent promising molecular targets for cancer treatment, we sought to characterize HDAC4 expression and function and its regulation by miR-29b in MM cells. HDAC4 protein levels and enzymatic activity were found up-regulated in a panel of 11 MM cell lines as compared to peripheral blood mononuclear cells from healthy donors. Moreover, the analysis of HDAC4 mRNA levels in a microarray dataset consisting of 4 healthy controls, 55 MM and 29 plasma cell leukemia patients indicated significant over-expression in cancer samples, suggesting that high HDAC4 expression might be involved in MM pathogenesis. Notably, the analysis of miRNA/mRNA paired expression in the same microarray dataset revealed an inverse correlation between miR-29b and HDAC4 mRNA, strengthening the relevance of miR-29b as a key regulator of HDAC4. Synthetic miR-29b mimics transfected in MM cells (SKMM1 and NCI-H929) down-regulated HDAC4 mRNA and protein levels and inhibited the 3’UTR of HDAC4 cloned in a luciferase reporter vector, whereas failed to regulate a 3’UTR devoid of two predicted miR-29b target sites. On the other hand, lentiviral-mediated overexpression of HDAC4 strongly inhibited miR-29b expression. These results underscore a negative feedback loop occurring between miR-29b and its target HDAC4 in MM cells. Through loss of function experiments, we assessed the functional significance of HDAC4 in MM cells. Stable silencing of HDAC4 by shRNAs induced growth inhibition, caspase 3/7-dependent apoptosis and autophagy in U266 and KMS11 cells, which occurred together to miR-29b up-regulation. Interestingly, the pan-HDAC inhibitor vorinostat also triggered apoptosis and autophagy in MM cells, along with the induction of miR-29b and the down-regulation of HDAC4 and other miR-29b-canonical targets like CDK6, MCL-1 and Sp1. miR-29b itself was able to promote autophagy, as assessed by beclin-1 up-regulation and LC3A/B proteolytic cleavage in miR-29b mimics-transfected MM cells, which was abrogated by constitutive expression of HDAC4. Finally, we provided evidence that miR-29b over-expression potentiated, whereas its stable inhibition dampened, apoptosis and autophagy triggered by vorinostat. In conclusion, our findings shed light on the oncogenic role of HDAC4, which can be targeted through miR-29b-based therapeutic approaches, and identify miR-29b as a relevant effector of vorinostat activity in MM cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Tumor Circulating Plasma Cells Detected By Flow Cytometric Single Platform Method Correlate with Clinical Response to Therapy and Unfavorable Patients' Characteristics

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4357-4357
Author(s):  
Vittorio Emanuele Muccio ◽  
Milena Gilestro ◽  
Elona Saraci ◽  
Andrea Capra ◽  
Alessandro Costa ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Clinical Response ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
Plasma Cells ◽  
Response To Therapy ◽  
Advisory Committees ◽  
Flow Cytometric ◽  
Baseline Characteristics

Background: In multiple myeloma (MM), different clinical parameters and molecular prognostic factors can predict disease course and response to therapy. The classification of myeloma patients includes laboratory parameters associated with higher tumor activity, resistance to therapy and proliferative competence. Tumor circulating plasma cells (TCPC) in MM patients showed a strong correlation with a more aggressive disease. Aim: For the first time, we quantified the amounts of TCPC with single platform flow cytometric method and evaluated their relationship with patients' baseline characteristics and response to therapy before maintenance. Methods: Whole peripheral blood samples from 413 newly diagnosed MM patients ≤65 years enrolled in the UNITO-MM-01/FORTE trial were collected. Patients were randomized [1:1:1; stratification: International Staging System (ISS) and age] to ARM A: carfilzomib-cyclophosphamide-dexamethasone (KCyd) followed by melphalan 200 mg/m2 and autologous stem-cell transplantation (MEL200-ASCT) and consolidation with 4 KCyd; ARM B: carfilzomib-lenalidomide-dexamethasone (KRd) followed by MEL200-ASCT and 4 KRd; ARM C: 12 KRd cycles. Enrollment was completed in March 2017; data cut-off was November 30, 2018. For the single platform tube, the antibody combination CD38PC7/CD138PC5.5/ CD45KO/CD56PE/CD19PB was mixed with 100µL of EDTA peripheral blood, dispensed with reverse pipetting, and incubated for 15 min, added with 500µL of lysing solution and, after 15 min, 100µL of flow count fluorospheres were dispensed with reverse pipetting and cells acquired with Navios flow cytometer. Intracytoplasmic tube was set up to confirm the clonality of CPC. Results: Circulating plasma cells (CPC) were quantified in 413 samples, with median values of 0.03% (range: 0-51%) and 2.37/mm3 (range: 0-6272/mm3). White blood cells were 5710/mm3 (range: 1752-26102/mm3); total events acquired 1285000 (range: 40000-2000000); median CPC events were 58 (range: 0-441000); cellular events acquired were 190000 (range: 4428-1300000). In 390 out of 413 samples (94.4%), CPC were detected; 272 samples (66%) showed TCPC with a median of 1.24/mm3 (range 0.06- 6272/mm3). Patients were sorted according to different baseline characteristics and the medians of absolute TCPC were compared. The most statistically significant differences (p<0.001) were: haemoglobin (Hb) <10 (12.9/mm3) vs. ≥10 (0.81/mm3); ISS I (0.30/mm3) vs. ISS II (2.85/mm3) vs. ISS III (5.14/mm3); R-ISS I (0.25/mm3) vs. II (2.76/mm3) vs. III (7.45/mm3); albumin <3.5g/dL (2.76/mm3) vs. ≥3.5g/dL (1.05/mm3); β2-microglobulin <3.5mg/dL (0.67/mm3) vs. 3.5mg/dL-5.5mg/dL (3.88/mm3) vs. >5.5mg/dL (16.47/mm3); lactate dehydrogenase (LDH) ≤upper limit of normal (ULN, 1.14/mm3) vs. >ULN (7.36/mm3); plasma cells (PC) in biopsy <60% (0.60/mm3) vs. ≥60%(2.76/mm3); with (3.18/mm3) vs. without amp1q (1.18/mm3); Morgan risk standard (1.21/mm3) vs. high (3.00/mm3). Finally, we compared the absolute number of TCPC and the quality of response at the end of consolidation therapy. Higher values of TCPC were related to worst response: <partial response (PR, 4.23/mm3) vs. ≥PR (1.23/mm3); <very good PR (VGPR, 2.91/mm3) vs. ≥VGPR (1.20/mm3); <complete response (CR, 1.95/mm3) vs. ≥CR (1.09/mm3); <stringent CR (sCR, 1.71/mm3) vs. ≥sCR (1.00/mm3), p<0.05. Conclusions: Single-platform flow cytometry is a simple method to quantify TCPC, present in almost all peripheral blood from MM patients; a high number is related to poor clinical response to therapy and helps in identifying high-risk patients. Moreover, it allows the discrimination between normal and pathological plasma cell population in peripheral blood. However, a longer follow up is needed to evaluate how TCPC can affect survival in patients with MM. Disclosures Musto: Amgen: Honoraria; Celgene: Honoraria. Gay:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Boccadoro:Sanofi: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; AbbVie: Honoraria; Mundipharma: Research Funding. Omedé:Janssen: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: This presentation includes discussion of off-label use of a drug or drugs for the treatment of multiple myeloma.

scholarly journals PIM1-Mediated CXCR4 Phosphorylation: A Potentially Class-Distinct Therapeutic Target of Next Generation Proteasome Inhibitors in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2774-2774
Author(s):  
Johannes M. Waldschmidt ◽  
Dagmar Wider ◽  
Stefan J. Müller ◽  
Anna Simon ◽  
Sarah Decker ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitor ◽  
Research Funding ◽  
Second Generation ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Protein Levels ◽  
Distinct Mechanism ◽  
Dose Dependent ◽  
Cxcr4 Axis

Abstract Introduction: The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment is fundamental to MM pathogenesis. Cell adhesion-mediated drug resistance (CAM-DR) is regulated by adhesion receptors on MM cells such as CXCR4, CXCR7, CD49d and CD44. We and others have previously reported that CAM-DR towards drugs like bortezomib, pomalidomide or vorinostat may be dissolved by combining these novel agents with the CXCR4 inhibitor plerixafor. Different than expected, additional treatment with plerixafor in corresponding experiment however did not rescue the cytotoxic effects of the second generation proteasome inhibitor carfilzomib. We hypothesized that carfilzomib itself interferes with the CXCR4-CXCL12 axis in myeloma. Prior reports in AML and CLL indicate that PIM1-mediated CXCR4 phosphorylation at the position S339 is an essential step for CXCR4 recirculation to the cell surface and its function as CXCL12 receptor (Grundler et al. 2009, Decker et al. 2014). In this project, we therefore examined the effects of carfilzomib on the PIM1-CXCR4 axis as a not yet described, potentially class-distinct mechanism of action of this second generation proteasome inhibitor. Methods: U266, RPMI-8226, L363, MOLT-4, NCI-H929 and the stromal cell line M2-10B4 were utilized. Bortezomib (1, 10, 20, 50, 100nM), carfilzomib (20, 50, 100nM) and plerixafor (10, 50, 100μM) were used based on previous studies and are well comparable to clinically relevant doses. CXCL12 stimulation was performed with human recombinant CXCL12 (30nM). For combination studies, cells were preincubated with plerixafor (50µM). Viability was quantified by propidium iodide and annexinV-FITC using flow cytometry. For quantitative real-time PCR and Western blots, U266 monocultured cells were treated with a carfilzomib pulse (t=1h), were allowed to recover for 20 hours, starved for 4 hours and stimulated with CXCL12 for 15 minutes (n=4). PIM-1 mRNA transcript levels were assessed in U266 control vs. U266 treated with a carfilzomib pulse (100nM, t=1h) by qPCR. Data was analyzed according to the "delta-delta-CT method" based on the relative expression of PIM-1 vs. GAPDH. Results were normalized to the mean of the control samples. Results: FACS analyses determined a substantial decrease of CD138 and CXCR4 surface expression in a dose-dependent manner after 1h carfilzomib treatment of U266 cells. Further assessment of downstream signaling revealed that carfilzomib treatment significantly reduces CXCR4 phosphorylation at S339 without changing total levels of CXCR4 (Figure A) or total levels of ERK or pERK (not shown), excluding a general inhibition of phosphorylation or protein synthesis by carfilzomib. Following the hypothesis that CXCR4 is potentially phosphorylated by PIM1 kinase, we assessed the impact of carfilzomib on PIM-1 protein levels: PIM-1 kinase protein was significantly reduced in a dose-dependent manner along with the levels of pCXCR4 in response to increasing doses of carfilzomib (0-100nM, Figure B). To further investigate a possible direct interference at the mRNA level, we evaluated PIM-1 mRNA levels after 1h carfilzomib, confirming substantially reduced PIM-1 RNA transcripts (Figure C). Different from carfilzomib and in line with prior observations (Shay et al. 2005), bortezomib was shown to increase protein levels of PIM-1 (data not shown). Side-by-side comparative assays of bortezomib vs. carfilzomib in terms of reduced CXCR4 expression, decreased CXCR4 phosphorylation and PIM-1 levels on mRNA and protein level are currently ongoing and will be presented at the meeting. Conclusions: Similar to previous reports on ixazomib reducing PIM-1 on protein and mRNA levels by inhibiting the tumor-suppressive microRNA miR33b (Tian et al. 2012), this work provides a potentially distinct mechanism of action of the second generation proteasome inhibitor carfilzomib on the PIM1-CXCR4 axis and identifies PIM-1 as a valid target to overcome CAM-DR in multiple myeloma. Figure Carfilzomib overcomes stroma protection due to PIM-1 kinase inhibition. Figure. Carfilzomib overcomes stroma protection due to PIM-1 kinase inhibition. Disclosures Engelhardt: Janssen: Research Funding; Amgen: Research Funding; MSD: Research Funding; Celgene: Research Funding.

scholarly journals Higher Intensity of Cell Surface Glucose-Regulated Protein 78 (csGRP78) Expression Is Seen in Patients with Early Progressive Disease/Mortality in a Cohort of Relapsed, Refractory Multiple Myeloma Patients Treated with Carfilzomib, Thalidomide and Dexamethasone

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4376-4376
Author(s):  
Slavisa Ninkovic ◽  
Simon Harrison ◽  
Lenny Straszkowski ◽  
Giulia Quattrocchi ◽  
Wee-Joo Chng ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Surface ◽  
Disease Progression ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Response To Therapy ◽  
Advisory Committees ◽  
Refractory Multiple Myeloma ◽  
Cell Surface Grp78

Background: GRP78, an endoplasmic reticulum stress-inducible molecular chaperone, is up-regulated at times of cellular stress to limit proteotoxicity and promote cell survival. Translocation of GRP78 to the cell surface (csGRP78) is emerging as a critical step providing tumour cells with a survival advantage. Here we quantified, monitored and correlated plasma cell (PC) csGRP78 expression in patients (pts) with relapsed/refractory multiple myeloma (RRMM) treated with carfilzomib, thalidomide and dexamethasone (KTd). Method: Patients enrolled in the single arm, multicentre, phase II Australasian Leukaemia & Lymphoma Group MM018/Asian Myeloma Network 002 study were treated with KTd as described previously (Quach et al. Blood 2018 132:1955). Formalin-fixed, paraffin-embedded BM trephine sections collected at baseline (n=29), after 6-months (mo) of KTd (n=19) and at time of disease progression (PD; n=5) were stained for CD138 and GRP78 by multiplex immunofluorescence histochemistry using the OpalTM workflow. Membrane expression of CD138 and GRP78 was extracted using inForm® software, compared across timepoints and correlated to disease characteristics and treatment outcomes. Descriptive statistics, paired/unpaired two-tailed t-test, Pearson's or Spearman's correlation were applied as appropriate. Results: Correlative BM biopsies were collected for 29 pts [male = 18, mean age = 65.0 years (range 41.9-83.2), 2 median prior lines of therapy (range 1-3)] at baseline, 21 pts after 6 months of KTd (7 had PD/died prior to cycle 6, 1 came off study due to grade 4 AE after 5 cycles) and 5 pts at time of PD. There was no difference in the number of fields, BM cellularity (%) or number of nucleated cells (NCs) assessed at baseline and 6mo (p=0.927, 0.331 and 0.491 respectively). PC burden (%; mean±SD) reduced significantly following 6mo KTd (28.3±28.1 vs. 2.12±2.37; p=0.0007). The number of plasma cells expressing csGRP78 (% of all NCs) was reduced following 6mo KTd treatment (27.04±26.83 vs. 2.05±2.32; p=0.0007) while the % of CD138-ve BM cells expressing csGRP78 (% of all NCs; mean±SD) increased (62.61±27.55 vs. 87.46±10.11; p=0.0005). Globally, there was a trend for reduced intensity of csGRP78 expression after 6mo KTd (H-score 70.79±62.16 vs. 53.53±51.45; p=0.2073). There was no correlation between baseline BM NC GRP78 H-score and baseline paraprotein level, involved/uninvolved serum free light chain ratio or depth of response to KTd. Pts with early (<6mo) disease progression/mortality (n=7) had a significantly higher baseline H-score (136±78.8 vs. 75.1±65.2; p=0.049). There was a separation of survival curves, but no significant difference regarding risk of early PD/mortality based a baseline H-score >75th percentile of the cohort (>152); p=0.1472 (Figure 1). Conclusion: Here we demonstrate that cell surface expression of GRP78 is prominent in both the plasma cells and cells of the tumour microenvironment in patients with RRMM and persists in the TME cells in patients on treatment. Early (<6mo) disease progression or mortality is associated with higher baseline intensity of global cell surface GRP78 expression. Additional studies are being performed to evaluate the promise of cell surface GRP78 expression on plasma cells as a potential biomarker of response to therapy. Disclosures Harrison: AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: investigator on studies, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Research Funding. Quach:Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees.

Inhibition of protein geranylgeranylation induces apoptosis in myeloma plasma cells by reducing Mcl-1 protein levels

Blood ◽  
2003 ◽  
Vol 102 (9) ◽  
pp. 3354-3362 ◽  
Author(s):  
Niels W. C. J. van de Donk ◽  
Marloes M. J. Kamphuis ◽  
Berris van Kessel ◽  
Henk M. Lokhorst ◽  
Andries C. Bloem
Keyword(s):  
Multiple Myeloma ◽  
Plasma Cells ◽  
Mevalonate Pathway ◽  
Cytochrome C Release ◽  
Hmg Coa Reductase ◽  
Myeloma Cells ◽  
Protein Levels ◽  
Geranylgeranyl Transferase ◽  
Induced Apoptosis ◽  
Coa Reductase

AbstractHMG-CoA reductase is the rate-limiting enzyme of the mevalonate pathway leading to the formation of cholesterol and isoprenoids such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). The inhibition of HMG-CoA reductase by lovastatin induced apoptosis in plasma cell lines and tumor cells from patients with multiple myeloma. Here we show that cotreatment with mevalonate or geranylgeranyl moieties, but not farnesyl groups, rescued myeloma cells from lovastatin-induced apoptosis. In addition, the inhibition of geranylgeranylation by specific inhibition of geranylgeranyl transferase I (GGTase I) induced the apoptosis of myeloma cells. Apoptosis triggered by the inhibition of geranylgeranylation was associated with reduction of Mcl-1 protein expression, collapse of the mitochondrial transmembrane potential, expression of the mitochondrial membrane protein 7A6, cytochrome c release from mitochondria into the cytosol, and stimulation of caspase-3 activity. These results imply that protein geranylgeranylation is critical for regulating myeloma tumor cell survival, possibly through regulating Mcl-1 expression. Our results show that pharmacologic agents such as lovastatin or GGTase inhibitors may be useful in the treatment of multiple myeloma.

scholarly journals Comparison of Minimal Residual Disease Detection between the Manual and Automated Gating Strategies of Euroflow Next-Generation Flow in Patients with Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3083-3083
Author(s):  
Hiroyuki Takamatsu ◽  
Takeshi Yoroidaka ◽  
Takeshi Yamashita ◽  
Ryoichi Murata ◽  
Mikio Ueda ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Partial Response ◽  
Pharmaceutical Company ◽  
Research Funding ◽  
Plasma Cells ◽  
Residual Disease ◽  
Next Generation ◽  
Chugai Pharmaceutical ◽  
Company Limited ◽  
Minimal Residual

Background: The rate of complete response (CR) in multiple myeloma (MM) has dramatically increased because of the development of novel agents. In addition, the development of methods for measuring minimal residual disease (MRD), such as multiparameter flow cytometry and next-generation sequencing, has made it possible to stratify CR patients according to the MRD extent. EuroFlow next-generation flow (EuroFlow-NGF) is considered one of the gold standard methods for evaluating the negative status of MRD in MM. The automated gating strategy of EuroFlow-NGF has been shown to detect MRD as accurately as the manual gating strategy by experts. Oberle et al. (Haematologica, 2017) have found that daratumumab persisted on the surface of myeloma cells treated with it and that the anti-CD38 multi-epitope antibody used in EuroFlow-NGF has partial cross-reactivity with daratumumab, leading to generally lower mean fluorescence intensities of CD38. Therefore, MRD levels may have been underestimated in patients who were treated with anti-CD38 monoclonal antibodies (mAbs) using the automated gating strategy, leading to inappropriate management of the patients. Because no studies have examined the correlation of MRD extent between the manual and automated gating strategies in patients with MM who have received anti-CD38 mAbs, we compared MRD detection between the two gating strategies of EuroFlow-NGF in patients with MM. Methods: The study included bone marrow samples from 51 patients with MM (27 male and 24 female patients), including 13 patients treated with anti-CD38 mAb (12 treated with daratumumab and 1 treated with isatuximab). The median patient age was 70 years (range, 32-92 years) at MRD assessment. The disease statuses at MRD assessment were stringent CR in 26 patients (51%), CR in 7 (14%), very good partial response in 13 (26%), partial response in 1 (2%), and progressive disease in 4 (8%). The sample preparation protocol, Ab panel, and automated gating strategy of EuroFlow-NGF have been reported previously (Flores-Montero et al. Leukemia 2017). Briefly, we performed the EuroFlow-NGF method, which involved ammonium chloride-based bulk lysis, followed by surface staining using antibodies against CD138-BV421, CD27-BV510, CD38 multiepitope (ME)-FITC, CD56-PE, CD45-PerCP Cy5.5, CD19-PECy7, CD117-APC, and CD81-APC C750 in tube 1 and surface/intracellular staining using antibodies against CD138-BV421, CD27-BV510, CD38 ME-FITC, CD56-PE, CD45-PerCP Cy5.5, CD19-PECy7, CD117-APC, CD81-APC C750, cytoplasmic (cy) Igκ-APC, and cyIgλ-APC C750 after permeabilization in tube 2. For data analysis, events from both eight-color tubes (tubes 1 and 2) were merged, and the values of all parameters per tube were mathematically calculated using the merge and calculation functions of Infinicyt software (Cytognos SL, Salamanca, Spain). Automatic identification and enumeration of total plasma cells (tPCs) and abnormal plasma cells (MRD) were performed using the automatic gating function of Infinicyt software as described previously (Flores-Montero et al. Leukemia 2017). We compared the total nucleated cell number, tPC ratio, and MRD ratio between the manual (by experts) and automated gating strategies of EuroFlow-NGF. Results: In patients with MM who did not receive any anti-CD38 mAb therapy, we observed high correlations for both the tPC (r = 0.959, P < 0.0001) (Figure A) and MRD (r = 0.974, P < 0.0001) (Figure B) ratios between the manual and automated gating strategies of EuroFlow-NGF. On the other hand, in patients with MM who received anti-CD38 mAb therapy, we did not observe good correlations for both the tPC (r = 0.349, P = 0.2) (Figure A) and MRD (r = 0.292, P = 0.3) (Figure B) ratios between the two strategies owing to a lower fluorescence intensity of CD38 on PCs. In addition, when the MRD threshold was set to 10-5, the discordance of MRD positivity/negativity between the two strategies was significantly higher in patients who received anti-CD38 mAb therapy than in those who did not receive anti-CD38 mAb therapy [4/13 (31%) vs. 1/38 (3%), P = 0.012]. Conclusion: Although the automated gating strategy of EuroFlow-NGF could be a viable alternative to the manual strategy for the assessment of MRD in MM, we may have to utilize the manual strategy to obtain precise MRD results for patients with MM who received anti-CD38 mAbs. Figure Disclosures Takamatsu: Celgene: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ono pharmaceutical: Honoraria, Research Funding; CSL Behring: Research Funding; SRL: Consultancy, Research Funding; Janssen Pharmaceutical: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Fujimoto Pharmaceutical: Honoraria; Becton, Dickinson and Company: Honoraria; Abbvie: Consultancy; Daiichi-Sankyo Company: Honoraria. Yoroidaka:Ono Pharmaceutical: Honoraria. Yamashita:Janssen Pharmaceutical K.K.: Honoraria; Daiichi-Sankyo Company: Honoraria; Kyowa Kirin: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; TEIJIN PHARMA LIMITED: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Bristol-Myers Squibb: Honoraria; Ono Pharmaceutical: Honoraria; Celgene: Honoraria. Murata:Celgene: Honoraria; Ono pharmaceutical: Honoraria. Nakao:Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy; Ohtsuka Pharmaceutical: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria. Matsue:Novartis Pharma K.K: Honoraria; Ono Pharmaceutical: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Janssen Pharmaceutical K.K.: Honoraria.

scholarly journals Targeting Cancer Associated Fibroblasts in the Bone Marrow Prevents Resistance to Chimeric Antigen Receptor T Cell Therapy in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 865-865 ◽  
Author(s):  
Reona Sakemura ◽  
Michelle J. Cox ◽  
Michael J. Hansen ◽  
Mehrdad Hefazi ◽  
Claudia Manriquez Roman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Therapy ◽  
Cell Line ◽  
Research Funding ◽  
Plasma Cells ◽  
Flow Cytometric Analysis ◽  
Data Safety Monitoring Board ◽  
Nsg Mice ◽  
Dual Targeting ◽  
The Impact

Cellular immunotherapy is a rapidly progressing field in multiple myeloma (MM). Multiple clinical trials have reported impressive efficacy of B cell maturation antigen (BCMA) directed chimeric antigen receptor cell therapy (BCMA CART) in MM. While trials demonstrated an overall response rate of 70-90% in patients with relapsed/refractory MM, the durable response rate is around 30%. Most patients lose their CART cells and the disease relapses within the first year, suggesting an inhibition by the MM tumor microenvironment (TME). Therefore strategies to overcome this inhibition would represent a major advance in CART cell therapy for MM. Cancer associated fibroblasts (CAFs) within the TME play a critical role in promoting tumor growth and in the generation of an immunosuppressive microenvironment. We hypothesized that CAFs from bone marrows of patients with MM (MM-CAFs) inhibit BCMA CART cells and contribute to their failure and that targeting both the malignant plasma cells and CAFs can overcome this resistance. To test this hypothesis, we isolated MM-CAFs and studied their interaction with BCMA CART cells generated from normal donors (41BB costimulated, lentivirally transduced). Our initial findings suggest that MM-CAFs inhibit BCMA CART cell antigen specific proliferation in the presence of the BCMA+ MM cell line OPM2, and this inhibition is predominantly mediated through the secretion of TGF-β (Fig A). MM-CAFs also promoted MM tumor growth in an MM-TME xenograft model established in the laboratory (Fig B). Here, immunocompromised NOD-SCID-γ-/- (NSG) mice were engrafted with 1x106 luciferase+ BCMA+ OPM2, in combination with either 1x106 CAFs or vehicle control intraveneously (IV). Subsequent tumor burden was monitored by bioluminescent imaging of these mice. The presence of CAFs in this model significantly accelerated MM progression (Fig B). Based on these findings, we aimed to develop CART cell therapy targeting both malignant MM cells and their CAFs and to determine whether this strategy can reverse MM-CAF induced CART cell inhibition. To identify targets for these CART cells, we first verified the expression of Fibroblast Associated Protein (FAP), an established CAF target, on MM-CAFs. Flow cytometric analysis of MM-CAFs showed significantly higher expression of FAP, compared to fibroblasts derived from normal bone marrow (Fig C). In addition, our screening flow cytometric analysis identified CS1 as another protein overexpressed by MM-CAFs (Fig C). We therefore designed and generated FAP CART cells (41BB costimulated, lentivirally transduced) and CS1 CART cells (CD28 costimulated, lentivirally transduced). We also generated dual CART cells for both BCMA-FAP CART cells and BCMA-CS1 CART cells. These dual CART cells were generated through the dual transduction of two lentiviral vectors during CART manufacturing. Next, we evaluated the impact of CAFs on effector functions of BCMA CART cells compared to dual targeting CART cells. When CART cells were stimulated with the BCMA+ MM cell line MM1S, in the presence of MM-CAFs, the antigen specific proliferation of BCMA CART cells, but not the dual targeting CART cells was significantly inhibited (Fig A). Similarly, in the presence of MM-CAFs, production of key effector cytokines by BCMA CART cells, but not the dual CART cells was reduced (Fig D). Finally, to verify the significance of our laboratory findings, we investigated the impact of CAFs on CART cell functions in vivo. First, using OPM2 xenografts, treatment with BCMA CART cells were able to completely eradicate MM (Fig E). However, to determine the effect of targeting CAFs, we used our MM-TME model. Here, NSG mice were engrafted with the luciferase+ MM cell line OPM2, along with MM-CAFs, as described in Fig 1B. Mice were then imaged for engraftment and randomized to treatment with 1) untransduced control T cells, 2) BCMA CART cells, 3) BCMA-FAP CART cells, or 4) BCMA-CS1 CART cells. A lower dose (1x106 IV) of CART cell was used to induce relapse post BCMA CART cells. Treatment with BCMA CART cells led to a transient antitumor activity in this MM-TME model (mice died within 2 weeks), while dual targeting CART cells resulted in durable remissions and long term survival of these mice (Fig F). In summary, we demonstrate for the first time that dual targeting both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART cell therapy in multiple myeloma. Figure Disclosures Sakemura: Humanigen: Patents & Royalties. Cox:Humanigen: Patents & Royalties. Parikh:Janssen: Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; AstraZeneca: Honoraria, Research Funding. Kay:Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Lentigen: Research Funding; Kite/Gilead: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Tolero: Research Funding; Novartis: Patents & Royalties, Research Funding; Morphosys: Research Funding.

Elimination of CD20-Expressing Cells in Multiple Myeloma by Iodine I-131 Tositumomab (Bexxar®) Correlates with Response to Therapy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5176-5176 ◽  
Author(s):  
Andrzej J. Jakubowiak ◽  
Malathi Hari ◽  
Tara Kendall ◽  
Yasser Khaled ◽  
Shin Mineishi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Stem Cell ◽  
Partial Response ◽  
Clinical Outcomes ◽  
Plasma Cells ◽  
Response To Therapy ◽  
Myeloma Cells ◽  
Consolidation Treatment ◽  
Anti Cd20

Abstract It has been proposed that treatment failures in multiple myeloma (MM) are related to the chemoresistance of a subset of malignant myeloma cells with clonogenic potential to the anti-myeloma drugs (Matsui et al., Cancer Research2008, 68:190). Studies suggest that these putative myeloma stem cells (MSC) are CD138neg and express CD20, which is usually absent in more differentiated malignant plasma cells (Matsui et al., Blood2004, 15:2332). These findings provided a rationale for a treatment strategy to eliminate chemoresistant clonogenic MSC using anti-CD20 antibodies. To evaluate the clinical effects of anti-CD20 treatment, we developed a phase II trial with Bexxar as a consolidation treatment for MM. We hypothesized that Bexxar would be more efficacious than unlabeled antibody in the eradication of highly radiosensitive myeloma cells by both direct and cross-fire effects. Preclinical studies showed that tositumomab, the antibody used in Bexxar, inhibited colony formation of clonogenic myeloma cells. To be eligible for Bexxar treatment, patients must have completed at least 4 cycles of therapy (1st to 3rd line) and have measurable disease in a plateau of at least partial response (PR). To date, 10 of 30 patients have been enrolled, of which 5 were treated with Bexxar after completion of initial therapy prior to proceeding to autologous stem cell transplant (ASCT). Patients proceeding to ASCT required hematopoietic stem cells collection prior to Bexxar and 3 months after Bexxar. Eight patients are evaluable for response and toxicities. At 3 months post Bexxar, 1 patient achieved a partial response (PR), 4 patients had stable disease (SD), and 1 patient progressed (PD). At 1 year post Bexxar, 1 patient with initial PR achieved CR and remains in CR, 1 patient is in unconfirmed CR, 2 are in partial response (PR), and 3 remain in SD. After a median 20 months of followup (range 4–24), all patient are alive, 4 in continued response, 3 with SD. Hematological toxicities were mild to moderate (1 patient grade 3 and 4 patients grade 2 thrombocytopenia, 4 patients grade 2 neutropenia). Non-hematological toxicity was limited to HAMA (6 patients). Out of patients who received Bexxar prior to transplant, 3 collected stem cells post-Bexxar without problems, one requires re-collection. Three patients who proceeded to ASCT to date using the post-Bexxar stem cell collection, engrafted at 11–12 days, and had no unexpected toxicities with ASCT. We also analyzed CD20+ cells in bone marrow aspirates (BM) and stem cell collections (SCC) using samples collected from 4 patients before and after Bexxar. Bexxar treatment eliminated a median 80% of CD20+ cells (range 23–97). For a given patient, elimination of CD20+ cells from SCC correlated with elimination of CD20+ cells from BM. The most complete elimination of CD20+ cells from BM was observed in 2 patients who at 1 year achieved CR (94% and 97%), compared to patients who achieved PR (23% and 68%). We conclude that anti-CD20 consolidation treatment of myeloma patients with Bexxar used as targeted therapy against clonogenic myeloma cells is feasible and well tolerated. Clinical outcomes to date are encouraging considering that clonogenic MSC represent &lt;5% of malignant plasma cells and delayed responses observed in this study could be expected. While early, clinical outcomes appear to correlate with the efficacy of the CD20+ cell elimination by Bexxar treatment in myeloma.

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