ALBERT

Description: Introduction: Oprozomib (OPZ) is an irreversible, orally administered proteasome inhibitor (PI).It decreases tumor burden and prevents tumor-related bone loss in preclinical multiple myeloma (MM) studies. In recently published clinical trials, the combination of the PI carfilzomib with the immunomodulatory agent (IMiD) lenalidomide and dexamethasone (Dex) has shown high response rates with durable responses for previously treated and untreated MM patients. Carfilzomib with the IMiD pomalidomide (Pom) and Dex for the treatment of RRMM patients shows promising results (Shah et al, 2013). We evaluated OPZ in combination with Pom and Dex using two of our human MM xenograft models in severe combined immunodeficient (SCID) mice. Methods: Each SCID mouse (n=10/group) was surgically implanted with a 20 - 40 mm3 MM tumor piece into the hind limb. Seven days post-implantation mice were randomized into treatment groups based on human immunoglobulin (Ig) G levels. OPZ stock solution (4 mg/ml) was diluted to 40 mg/kg using 1% carboxymethylcellulose (CMC) and administered twice weekly on two consecutive days via oral gavage.Dex stock solution (10 mg/ml) was diluted to 1 mg/kg using NaCl and administered daily via intraperitoneal injection.Pom stock solution (1 mg/ml) was diluted to 10 mg/kg using 1% CMC and administered daily via oral gavage. Tumor size was measured using calipers and IgG levels by ELISA. Results: Using our human MM model LAGk-1A, treatment with single agent OPZ or Pom produced a minimal reduction in tumor volume when compared with vehicle-treated mice, whereas Dex alone or OPZ + Pom produced more anti-MM effects, and no differences were observed between these two groups. Mice treated with OPZ + Dex or Pom + Dex also showed greater anti-MM activity than OPZ + Pom or Dex alone but the differences were not significant. All three agents together resulted in much smaller tumors when compared to OPZ + Pom on days 35, 42, 49 and 56 (P = 0.0006, P = 0.0001, P = 0.0002 and P 〈 0.0001, respectively). The same triplicate resulted in a smaller tumors when compared to OPZ + Dex on days 35, 42, 49, 56, and 63 (P = 0.0112, P = 0.0030, P = 0.0060, P = 0.0035 and P = 0.0021, respectively). Although Pom + Dex had some anti-MM effects when compared to the three single agents and one of the doublets (OPZ + Pom), mice receiving the triplicate demonstrated markedly smaller tumors when compared with Pom + Dex on days 35, 42, 49, 56, 63, 70, and 77 (P = 0.0250, P = 0.0018, P 〈 0.0001, P = 0.0014, P = 0.0018, P = 0.0017 and P = 0.0014, respectively). Mice receiving Pom + Dex had to be euthanized on day 77, whereas mice receiving all three drugs had very small tumors at study termination. We obtained similar results in a second MM xenograft model that produces IgG (LAGλ-1). Although day 21 post-tumor implantation mice receiving Pom alone had lower IgG levels compared with vehicle-treated mice (P = 0.0053), mice receiving OPZ + Pom had smaller tumors when compared to Pom alone (P = 0.0387), OPZ alone (P = 0.0004), or vehicle-treated mice (P = 0.0001). Although mice receiving Pom alone had a reduction in tumor size when compared with vehicle-treated mice (P = 0.0021), mice receiving OPZ + Pom had smaller tumors when compared with Pom alone (P = 0.0081), OPZ alone (P = 0.0007), or vehicle-treated mice (P 〈 0.0001). Mice receiving the triplicate showed smaller tumors on day 28 compared with mice treated with either the doublets of Pom + Dex (P = 0.0120) or OPZ + Pom (P = 0.0043). Conclusions: These in vivo human MM xenograft studies show that the combination of OPZ + Pom + Dex shows greater anti-MM activity than doublets (Pom + Dex, OPZ + Dex or OPZ + Pom) or single agents, and provides further support for this three drug combination to treat MM. Disclosures Tang: Onyx: Research Funding. Berenson@imbcr.org:Onyx: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Description: Introduction: A hallmark of multiple myeloma (MM) is the low levels of uninvolved immunoglobulin (Ig) levels. B-cell maturation antigen (BCMA) is a receptor expressed in mature non-malignant and malignant B lymphocytes, including plasma cells. Its ligands are B-cell activating factor (BAFF) and a proliferation inducing ligand (APRIL). We previously demonstrated that BCMA is present in the serum of MM patients (pts) and that its levels predict survival (Sanchez et al. Br J Haematol 2012). We hypothesized that circulating BCMA binds it ligands, preventing normal plasma cell development in MM patients which may explain their reduction in uninvolved Ig levels. Methods: BCMA-Fc and control Ig were obtained and reconstituted in PBS (R&D Systems). Retro-orbital bleeds were performed on C57 Bl/6 and SCID mice implanted with the human MM xenografts LAGλ-1, LAGk-1A or LAGk-2. Human BCMA and mouse BAFF, IgM, IgA and IgG levels were measured with ELISA (R&D Systems & Bethyl Laboratories). The Raji B-cell line was obtained from the American Type Culture Collection (Rockville, MD, USA). Human IgA and IgG levels were determined in MM patients using nephelometry (Immage 800, Beckman Coulter). Hevylite® Assays (Binding Site) were used to quantify the levels of heavy-light chain isoform pairs in MM patients. Results: We determined if mBAFF formed complexes with human BCMA (hBCMA) in the plasma from SCID mice implanted with LAGλ-1, LAGκ-2 or LAGκ-1A, and were able to identify mBAFF-hBCMA complexes in plasma samples from these mice. To determine what effect human BCMA had on Ig levels in immune competent mice, rhBCMA-Fc or control Ig-Fc (100 mg) was injected into C57 Bl/6 mice, and plasma IgA, IgM and IgG levels were measured. Decreases in IgA levels were observed following BCMA treatment when compared to baseline plasma IgA on days 4 and 6 (P = 0.0031 and P = 0.0064, respectively), and the control group (P = 0.0087 and P = 0.0221). Samples were also analyzed for mouse IgM levels with similar marked reductions when compared to the untreated (P = 0.0001) and Ig-Fc (P = 0.0088) groups. For plasma IgG levels, a marked decrease was observed on day 6 following rhBCMA-Fc administration when compared to its baseline levels (P = 0.0023), and also when compared to the Ig-Fc control protein (P = 0.0014) and the untreated control (P = 0.0129) groups. We then set out to determine if sera from MM patients contained BCMA-BAFF complexes, using ELISA plates coated with an anti-human BAFF antibody followed by exposure to a polyclonal anti-human BCMA antibody. A strong absorbance indicating the presence of BCMA-BAFF complexes was detected in serum samples from MM patients, whereas no antibody cross reactivity was observed in control samples. We also determined whether human MM serum or rhBCMA-Fc blocked BAFF from binding to Raji B-cells. Raji cells (B-cell line) were incubated with serum from a MM patient containing hBCMA (0.75μg/ml) or rhBCMA-Fc (3 μg/ml) in the presence of rhBAFF (500 ng/ml). Myeloma serum and rhBCMA-Fc decreased rhBAFF binding to Raji cells by 71% (from 96.8 to 25.6 %) and 74% (from 96.8 to 22.9 %), respectively. Next, we determined whether serum BCMA levels inversely correlated with uninvolved Ig levels in MM pts. For pts with IgA (n = 134) or IgG (n = 313) MM, higher BCMA levels (〉 100 ng/ml) correlated with below normal levels of uninvolved IgG in IgA MM and uninvolved IgA in IgG MM, whereas lower BCMA levels (〈 100 ng/ml) correlated with normal uninvolved levels (P 〈 0.0001). Using the Hevylite Assay, similar results were observed for the levels of BCMA compared to uninvolved IgG isoforms in both pts with involved IgG lambda (n = 62, P = 0.0006) and IgG kappa (n = 117, P 〈 0.0001) MM. Conclusions: Our laboratory previously has reported that serum levels of BCMA are increased in the serum of MM patients, and correlates with response to treatment and predicts survival. We now demonstrate 1) the formation of complexes of BCMA with its B-cell ligand BAFF in the plasma of MM xenografts and sera of MM patients, 2) show that rhBCMA and MM patient serum blocks the binding of BAFF to human B-cells, 3) administration of rhBCMA to normal mice results in marked reductions in their antibody levels, and 4) show that BCMA levels inversely correlate with uninvolved Ig levels in MM pts. Thus, the lack of normal antibody production in MM pts results, in part, from circulating BCMA binding its ligands, preventing production of normal antibody-producing cells. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: The proteasome inhibitor (PI) bortezomib is a novel anticancer drug that shows activity especially for treating patients with multiple myeloma (MM). However, its clinical efficacy has been hampered by the emergence of drug-resistance phenomena, the genetic and molecular basis of which remains elusive. We propose that the resistance to bortezomib treatment may be related to a polymorphism in a gene known to be a target for bortezomib's activity which has been identified as CIP2A which is a tumor suppressor PP2A inhibitor. We have used a high resolution melting curve (HRM) approach to screen several CIP2A SNPs containing significant genetic changes that may be different between bortezomib sensitive and resistant MM patients. In this study, we evaluated SNPs in this gene in healthy subjects and MM patients who were sensitive or resistant to bortezomib-based therapy. Methods: A panel of DNA samples was purified from MM patients (N=73) and healthy subjects (N=95). The samples were divided into three groups: bortezomib sensitive MM patients (N=61), bortezomib resistant MM patients (N=12), and healthy subjects. A single nucleotide polymorphism (SNP) of the bortezomib target genes CIP2A was selected. The PCR prime pairs for each SNP marker were designed with the software "Primer Expression 3" (Thermo Fisher Scientific). The genotype of each DNA sample was determined with the high resolution melt curve (HRM) technology on a real-time PCR instrument, StepOne Plus (Thermo Fisher Scientific). Results: The frequency of heterozygosity and homozygosity of CIP2A, SNP rs34172460(S258A) was 0.049 (3 out of 61 cases) in the bortezomib sensitive group, while it was 0.25 (3 out of 12 cases) in the bortezomib resistant group, a 5-fold increase in the frequency of this SNP. The frequency of heterozygosity and homozygosity of this same SNP in the healthy subjects was only 0.03 (3 out of 95 samples).The minor allele frequency (MAF) in the normal population is 0.046, according to the SNP data base, compatible with what we observed in both our healthy subjects and the bortezomib sensitive MM patients. Conclusion: This preliminary data suggests that heterozygotic and homozygotic mutants of rs34172460(S258A) may have an impact on resistance to bortezomib among patients with multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Description: Background: Waldenström's macroglobulinemia (WM) is an incurable B-cell lymphoplasmacytic lymphoma. B-cell maturation antigen (BCMA) serves as one of the receptors for B-cell activating factor (BAFF) or a proliferation-inducing ligand (APRIL), which are members of the tumor necrosis factor (TNF) family that can induce activation of NF-κB and promote survival of B cells, including neoplastic B cells. We previously showed that serum BCMA levels are elevated in multiple myeloma (MM) patients, and correlated with disease status and overall survival (OS). In this study, we sought to determine whether BCMA levels are elevated in the serum of WM patients, track with conventional WM tumor markers, and correlate with disease status and OS. Methods: Data was obtained on a total of 67 WM patients who received treatment in one of two clinics that specialize in the treatment of WM. Serum BCMA levels were determined with a polyclonal anti-BCMA antibody using an ELISA (R&D Systems, Minneapolis, MN). Mann-Whitney analysis was used to measure differences in serum BCMA levels between WM patients and healthy subjects as well as the clinical status of WM patients. Using Kaplan-Meier analysis, OS (median follow up 5.1 years) in WM patients was measured from the time of the first assessment of serum BCMA levels in each patient. In addition, serum BCMA levels were compared to serum M-protein and IgM levels in 12 patients, serially, during their individual courses of treatment. Results: We compared serum BCMA levels for all 67 patients, including 27 untreated WM patients to healthy subjects (n = 76). Serum BCMA levels were markedly elevated in all WM patients (median 79.53 ng/mL), including those who were untreated (median 82.72 ng/mL) versus healthy subjects (median 6.32 ng/mL, p 〈 0.0001 for both comparisons). Serum BCMA values were compared to both serum M-protein and IgM levels in 12 WM patients during their disease course. Serum BCMA levels consistently correlated with changes in both of these established WM serum markers during their course of disease. Serum BCMA levels of WM patients also correlated with patient disease status at the time of sample collection. Specifically, serum from patients achieving 〉 partial response (PR, n = 12) showed significantly lower levels of BCMA than samples from patients with either stable disease (SD, n = 8, p = 0.030) or progressive disease (PD, n = 21, p = 0.0004). OS (median 12.5 years) was determined among patients in the highest serum BCMA quartile (n=16; quartile range 128.91-812.48 ng/mL) and compared to levels in the lower three quartiles (n = 48; range 20.13-128.85 ng/mL). Notably, OS was shorter among patients in the top quartile compared to those in the other three quartiles (p = 0.02). Conclusions: This is the first study to demonstrate that serum BCMA levels are elevated in Waldenström's macroglobulinemia patients. Importantly, serum BCMA levels correlated with both serum IgM and M-protein levels, as well as tracked with these established WM biomarkers for individual patients during their course of disease. Additionally, patients with levels of BCMA in the highest quartile exhibited shorter OS. Thus, serum BCMA represents a new serum biomarker to predict outcome and monitor patients with Waldenström's macroglobulinemia. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: The bone marrow (BM) microenvironment plays an important role in multiple myeloma (MM). The BM niche is composed of multiple cell types including macrophages. Macrophages polarize into pro-inflammatory macrophage-1 (M1) or alternative M2 states that promote tumor growth and metastasis. We evaluated the proportion of M2 macrophages in BM from MM pts either showing complete response (CR) or progressive disease (PD), the effects of MM cells on M1 and M2 differentiation, and the role of Trib1 in M2 differentiation in MM BM. Since the JAK-STAT signaling pathway plays key roles in macrophages, we also evaluated the effects of the JAK2 inhibitor ruxolitinib (RUX) on M2 polarization in MM. Methods: Using immunofluorescence (IFC), we determined the proportion of M1 and M2 macrophages in BM biopsies and aspirates from MM pts with PD or CR. The BM biopsy samples were stained with antibodies directed against human iNOS and CD86 for M1 and arginase 1(ARG1) and CD36 for M2 cells. MM BM aspirates were also examined using flow cytometric analysis (FCA). Human monocytes isolated from healthy subjects or the THP1 monocyte cell line were co-cultured with MM cell lines (RPMI8226 and U266) or primary MM tumor cells. The effects of RUX at low concentrations (IC20) on M2 polarization were determined. The percentages of M1 and M2 macrophages were determined using FCA. Total RNA was extracted from monocytes. Quantitative PCR was measured with TaqMan technology. For the in vivo studies, human MM tumors (LAGκ-2) were surgically implanted into the left superficial gluteal muscle of SCID mice and tumor volume measured on a weekly basis. Results: The proportion of M2 macrophages (CD36+/ARG1+) was markedly increased in BM biopsies or mononuclear cells from MM pts with PD compared with those in CR using IFC staining. FCA also showed the percentage of M2 macrophages in BM was significantly increased in MM pts with PD (n=25) compared to those in CR (n=10; P=0.005) whereas there was no difference in the percentage of M1 (CD86+/iNOS+) macrophages in BM derived from MM pts with PD compared to those in CR. Trib1 gene mRNA levels were higher among pts with PD compared to those in CR whereas the gene expression of Trib2 and Trib3 was not different. Next, we co-cultured MM cell lines (U266) or fresh MM BMMCs with purified healthy human monocytes for one week. The percentage of M2 cells markedly increased and the proportion of M1 cells decreased. Trib1 gene expression increased during co-culture whereas there was no change in expression of the other two Tribs. When direct cell-to-cell contact occurred between the MM tumor cells and the monocytes, the percentage of M2 macrophages markedly increased. We investigated the effects of the JAK2 inhibitor RUX on M2 differentiation induced with MM tumor cells. After exposure to a low concentration of RUX, the percentage of M2 cells decreased when the monocytes were co-cultured with MM tumor cells. Trib1 gene expression of the monocytes treated with RUX was also notably reduced compared with cells not treated with the JAK2 inhibitor. Using our human MM xenograft model LAGκ-2, RUX (1.5mg/kg) reduced tumor growth and decreased the proportion of M2 macrophages in the tumor tissue of MM tumor-bearing SCID mice. Conclusion: M2 cells are present at high levels in BM derived from MM pts with PD compared to those in CR, MM cells induce monocytes to become M2 macrophages and increase Trib1 gene expression. This induces monocyte differentiation into M2 macrophages that support MM tumor cell growth.. Notably, the JAK2 inhibitor RUX inhibits both M2 macrophage polarization and Trib1 gene expression in MM, and reduces tumor growth in SCID mice bearing human MM. These results suggest that RUX may be effective for treating MM pts. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: The levels of serum monoclonal immunoglobulins (M-Igs) are used to monitor multiple myeloma (MM) patients. However, these assessments do not discriminate between normal polyclonal immunoglobulins (uninvolved) and M-Igs since they cannot determine the type of light chain associated with each immunoglobulin class (i.e. IgGκ, IgGλ, IgAκ, IgAλ, IgMκ, and IgMλ). The HevyLite® +(HLC) assays are able to accomplish this but the usefulness of these results for MM patients needs to be further established. We evaluated the levels of involved and uninvolved HLC levels, their ratios and differences and their relationship to outcomes among MM patients. Materials and Methods: Serum samples (n=189) from MM patients were analyzed using the HLC assays. Manufacturer’s HLC normal reference ranges were used. HLC results were correlated with clinical status as determined at the time of sampling and divided into groups according to clinical status (complete response (CR), ≥ partial response (PR) , 〈 partial response, and progressive disease (PD)). Normality was assessed using the D’Agostino-Pearson omnibus normality test. Statistical comparisons were made using t-student’s or Mann-Whitney tests as appropriate as well as Fisher’s test. Progression-free survival (PFS) was calculated using Kaplan--Meier analysis for specific regimens received during the time the samples were taken. All tests were double-tailed and p-values lower than 0.05 were considered to be statistically significant. Results: All MM serum samples analyzed had IgG (62%) or IgA (38%) isotypes. Results from the involved HLC/uninvolved HLC ratios and their differences demonstrated that samples from patients with PD had significantly both higher ratios and differences (P

Description: Introduction: The JAK2 inhibitor ruxolitinib (RUX) is an inhibitor of the Janus kinase family of protein tyrosine kinases (JAKs) that is effective for the treatment of myeloproliferative diseases. Immunomodulatory drugs (IMiDs) including lenalidomide (LEN) and corticosteroids have shown efficacy for the treatment of multiple myeloma (MM). The JAK-STAT signaling pathway plays key roles in the growth and survival of malignant plasma cells in MM. In this study, we evaluated the preclinical anti-MM effects of RUX in combination with LEN and corticosteroids, both in vitro and in vivo, and in a patient with MM and polycythemia rubra vera (PRV). Methods: The human MM cell lines U266, RPMI8226 and MM1S cells were derived from ATCC. Primary MM tumor cells were isolated from MM patients’ bone marrow aspirates. The cells were seeded at105 cells/100ul/well in 96-well plates and incubated for 24 h in the presence of vehicle, RUX, LEN or dexamethasone (DEX) alone, RUX + LEN, RUX + DEX, or all three drugs together for 48 h. Cell viability was quantified using the MTS cell proliferation assay. In vitro, synergy between ruxolitinib and lenalidomide or dexamethasone was assessed using the median effect method of Chou and Talalay. For the in vivo studies, the human myeloma tumors (LAGκ-1A or LAGκ-2) were surgically implanted into the left superficial gluteal muscle of anaesthetized naive SCID mice. Mice were blindly assigned to one of the experimental groups, and treatment was initiated 7–21 d after tumor implantation. LEN was administered via oral gavage daily (30 mg/kg). RUX (3 mg/kg) was given via intraperitoneal (IP) injection twice daily. Dexamethasone was administered daily (1.5mg/kg) via IP injection. An 88 year old MM patient with PRV who developed MM on RUX alone and then progressed on LEN+DEX was treated with the combination of all three drugs. Results: In vitro, RUX induced concentration-dependent inhibition of viability in all three MM cell lines (U266, RPMI8226 and MM1S) at RUX 50 mM and inhibition of primary MM tumor cells at a higher concentration (100 mM). In contrast, RUX had negligible cytotoxic effects on normal peripheral blood mononuclear cells (PBMCs). We next examined cell viability in the presence of RUX plus LEN or DEX. First, U266 cells were incubated with a fixed concentration of LEN (30 mM) or DEX (40 mM) with increasing concentrations of RUX (0.1–100 mM) for 48 h. At RUX 50 mM, the cytotoxic effects of LEN were enhanced and at RUX 1 mM, the anti-myeloma effect of DEX was increased. Moreover, the cytotoxic effects of RUX, LEN and DEX were greater than RUX in combination with either LEN or DEX in U266 cells. Similar results were obtained using the RPMI8226 and MM1S cell lines as well as primary MM tumor cells. Next, we evaluated RUX in combination with lenalidomide and dexamethasone in vivo using SCID mice bearing either the human LAGκ-1A or LAGκ-2 MM xenografts. RUX (3mg/kg), LEN (15mg/kg) or DEX (1mg/kg) alone did not inhibit tumor growth in either mice bearing LAGκ-1A or LAGκ-2. In contrast, the combination of RUX with DEX but not LEN slightly decreased tumor volume. However, the combination of all three drugs at the same doses showed a marked reduction of tumor size and delay of tumor growth in both human MM xenograft models. In addition, a patient with MM and PRV experienced sustained and ongoing reductions in his serum M-protein, IgG, and 24-urine M-protein with achievement of a partial response on low doses of RUX (2.5 mg twice daily), LEN (2.5 mg daily), and methylprednisolone (20 mg daily) that has been ongoing for more than 12 months after developing MM on RUX alone and then progressing on the combination of LEN and methylprednisolone. Conclusion: This study illustrates that the combination of the JAK2 inhibitor RUX, LEN and corticosteroids shows both preclinical and promising clinical results for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: We previously demonstrated that when carfilzomib (CFZ) was administered first to SCID mice bearing our human multiple myeloma (MM) xenografts, followed by the addition of melphalan (MEL) at the time of disease progression, a modest further reduction in tumor size was observed compared with continuing CFZ or adding MEL alone. In addition, substitution of single agent bortezomib (BOR) for CFZ showed no anti-MM effects. However, when MEL was administered first and CFZ was added after disease progression, mice treated with the combination showed a reduction in tumor volume compared with mice that discontinued MEL. Mice treated with CFZ alone showed no reduction in tumor size. Throughout the study, there was a trend toward smaller tumors among mice in which MEL was followed by the addition of CFZ when compared to mice in which MEL was continued or discontinued and treated with single-agent CFZ or vehicle alone. In the current study, we evaluated the anti-MM effects of CFZ, MEL and BOR for severe combined immunodeficient (SCID) mice progressing from BOR+MEL treatment using our human MM xenograft model LAGk-1A. Methods: Each SCID mouse was surgically implanted with a 20 – 40 mm3 LAGk-1A tumor piece into the left hind limb muscle. Seven days post-implantation mice were randomized into treatment groups based on human immunoglobulin (Ig) G levels. Carfilzomib stock solution (2 mg/ml) was diluted to 3 mg/kg using 5% dextrose and administered twice weekly on two consecutive days (Sundays and Mondays) via intravenous (i.v.) injection.Bortezomib stock solution (1 mg/ml) was diluted to 0.25 mg/kg using NaCl and administered twice weekly (Sundays and Tuesdays) via i.v. injection.MEL stock solution (3 mg/ml) was diluted to 1 mg/kg using PBS and administered once weekly (Fridays) via intraperitoneal injection. Mice (n = 10/group) were initially treated with the combination of BOR + MEL until tumor progression. Progression was defined as an increase in paraprotein 〉 25% confirmed on one consecutive assessment. After tumor progression, mice initially treated with BOR + MEL were randomized to continue to receive BOR + MEL, receive MEL only (discontinue BOR), receive BOR only (discontinue MEL), substitute BOR with CFZ, discontinue BOR + MEL and add in CFZ alone, or discontinue treatment altogether. Tumor size was measured using standard calipers and human IgG levels with an ELISA (Bethyl Laboratories, Montgomery, TX). This study was conducted according to protocols approved by the Institutional Animal Care and Use Committee. Results: Following progression from the combination of BOR + MEL among mice bearing LAGk-1A, substitution of these drugs with single agent CFZ alone did not produce a reduction in tumor volume when compared with mice continuing to receive BOR + MEL. However, significantly smaller tumors were observed when mice progressing from BOR + MEL were switched to CFZ + MEL compared with mice continued on BOR alone (P = 0.0044) or animals switched to CFZ alone (P = 0.05). There was a trend throughout the study toward smaller tumors in mice receiving CFZ + MEL when compared to mice continuing on BOR + MEL, receiving BOR, CFZ or vehicle alone or discontinuing BOR + MEL. Following progression from BOR + MEL, similar anti-MM effects were observed on human IgG (paraprotein) levels among animals treated with CFZ + MEL when compared to mice continuing BOR + MEL, receiving BOR alone switching to CFZ alone or discontinuing both BOR and MEL. Conclusions: These in vivo studies using our human MM xenograft model, LAGk-1A, show that SCID mice progressing from initial BOR + MEL treatment show a reduction in MM tumor burden when BOR is replaced with CFZ at the time of disease progression, and these tumors are significantly smaller than among mice continued on BOR + MEL. These studies demonstrate that once tumors progress from BOR + MEL treatment, CFZ can replace BOR and produce anti-tumor effects. A recent clinical trial confirms our preclinical findings and shows that most MM patients refractory to BOR in combination with other agents, including MEL, will respond to the substitution of CFZ for BOR (Berenson et al., Leukemia 2014). Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Macrophages polarize into pro-inflammatory M1 or alternative M2 states with distinct phenotypes and physiological functions. M2 cells promote tumor growth and metastasis through secretion of growth factors. However, crosstalk between tumor cells and macrophages in the development of M2 polarization has not been well demonstrated. We evaluated the proportion of M2 macrophages in bone marrow (BM) from patients (pts) with multiple myeloma (MM), the effects of MM cells on M1 and M2 differentiation, and the role of Trib1 in M2 differentiation in MM BM. Since the JAK-STAT signaling pathway plays key roles in the cell growth and differentiation of macrophages, we evaluated the effects of the JAK2 inhibitor ruxolitinib (RUX) on M2 polarization in MM. Methods: Using immunofluorescence (IFC) analysis, we determined the proportion of M1 and M2 macrophages in BM biopsies from MM pts with progressive disease and in remission. The BM biopsy samples were stained with antibodies directed against human iNOS and CD86 for M1 and arginase 1(ARG1) and CD36 for M2 cells, following a standard IFC protocol. Monocyte/macrophage phenotypes for M1 and M2 subtypes in mononuclear cells isolated from MM BM aspirates were also examined using flow cytometric analysis with these same antibodies. Human monocytes isolated from normal subjects or the THP1 monocyte cell line were co-cultured with MM cell lines or primary MM tumor cells with or without exposure to low concentrations (IC20) of ruxolitinib (RUX) using Transwell plates. The percentage of M1 and M2 macrophages was determined using flow cytometric analysis. Total RNA was extracted from monocytes followed the manufacturer’s directions. Quantitation PCR were measured with TaqMan technology performed in an OneStepPlus instrument. Results: IFC demonstrated that the percentage of M2 macrophages (CD36+/ARG1+) was markedly increased in BM sections from MM pts with progressive disease compared with those pts in remission. The flow cytometric data also showed the percentage of M2 (CD36+/ ARG1+) macrophages in BM was significantly increased in MM patients with progressive disease (n=20) compared to those in remission (n=9; P=0.005) whereas there was no significant difference in the percentage of M1 (CD86+/iNOS+) macrophages in BM derived from MM patients with progressive disease compared to those in remission. The results of both RT-PCR and Quantitation PCR showed Trib1 gene expression levels were higher among patients with progressive disease compared to those in remission. In contrast, the gene expression of Trib2 and Trib3 was not related to the MM patient’s clinical status. To determine whether MM tumor cells affected monocyte/macrophage differentiation and Trib gene expression, we co-cultured MM cell lines or fresh MM tumor cells with purified healthy human monocytes using Transwell plates. The percentage of M2 cells markedly increased whereas the proportion of M1 cells decreased. The expression of Trib1 increased during the 7 days of co-culture whereas there was no change in the expression of Trib2 and Trib3. Moreover, when direct cell-to-cell contact occurred between the MM cells and the monocytes, the percentage of M2 macrophages markedly increased after 7 days of incubation. It has been reported that Trib-1 mediated regulation of the MAPK/ERK pathway in a murine model and the Ras/Raf-1/MEK1/ERK cascade culminates in up-regulated expression of the gene encoding STAT3 whereas recruitment and activation of tyrosine kinase JAK-2 phosphorylates it. Thus, we investigated the effects of the JAK2 inhibitor RUX on M2 differentiation induced with MM tumor cells. The percentage of M2 cells was decreased when the monocytes that were co-cultured with MM tumor cells were treated with a low concentration (IC20) of RUX. Trib1 gene expression of the monocytes treated with RUX was also reduced comparing to the cells untreated with JAK2 inhibitor. Conclusion: Our results show that MM cells induce monocytes to become M2 macrophages and increase Trib1 gene expression providing a positive feedback loop on Trib1 expression, monocyte differentiation and tumor cell growth. M2 cells are present at high levels in BM derived from MM pts with progressive disease compared to those in remission. Notably, the JAK2 inhibitor RUX shows inhibition of both M2 macrophage polarization and Trib1 gene expression in MM, and these results suggest this drug may be effective for treating MM. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: A hallmark of multiple myeloma (MM) is the low levels of uninvolved immunoglobulin (Ig) levels. B-cell maturation antigen (BCMA) is a receptor expressed in mature non-malignant and malignant B lymphocytes, including plasma cells. Its ligands are BAFF (B-cell activating factor) and APRIL (a proliferation inducing ligand). We previously demonstrated that BCMA is present in the serum of MM patients (pts) and that its levels predict survival (Sanchez et al. Br J Haematol 2012). We hypothesized that circulating BCMA binds it ligands, preventing normal plasma cell development in MM patients which may explain their reduction in uninvolved Ig levels. Methods: BCMA-Fc and control Ig were obtained and reconstituted in PBS (R&D Systems). Retro-orbital bleeds were performed on SCID mice implanted with the human xenografts LAGλ-1, LAGk-1A or LAGk-2 following BCMA treatment. Human BCMA and mouse BAFF, IgM and IgA levels were measured with ELISA (R&D Systems & Bethyl Laboratories). Human IgA and IgG levels were determined in MM patients using nephelometry (Immage 800, Beckman Coulter). Hevylite® Assays (Binding Site) were used to quantify the levels of heavy-light chain isoform pairs in MM patients. Results: Recombinant mouse BAFF (rmBAFF) was mixed with recombinant human BCMA (rhBCMA) and incubated on plates coated with anti-mouse BAFF antibody (Ab). An anti-BCMA detection Ab was added, and BCMA-BAFF complexes were easily detected at concentrations of human BCMA and BAFF present in MM pts. We determined if mBAFF from SCID mice would also bind rhBCMA. SCID mice were bled; their plasma isolated and incubated with rhBCMA, and showed that rhBCMA formed complexes with mBAFF. To determine what effect human BCMA had on Ig levels in immune competent mice, rhBCMA-Fc or control Ig-Fc (100 mg) was injected into C57 Bl and Balb/c mice, and plasma IgA and IgM levels were measured. Following rhBCMA-Fc injection, a marked decrease in both antibody classes was observed in both mouse strains. Decreases in IgA levels were observed following BCMA treatment when compared to baseline plasma IgA on days 4 and 6 (P = 0.0031 and P = 0.0064, respectively), and the control groups (P = 0.0087 and P = 0.0221). Samples were also analyzed for mouse IgM levels with similar marked reductions when compared to the untreated (P = 0.0001) and Ig-Fc (P = 0.0088) groups. To determine if rhBCMA-mBAFF complexes formed in vivo, an ELISA was performed. Plates were pre-coated with a monoclonal mouse anti-BAFF capture Ab. Plasma samples were incubated and an anti-human-BCMA detection Ab was added. rhBCMA-mBAFF complexes were detected at high levels in plasma samples from mice dosed 4 and 6 days previously with BCMA-Fc whereas no complexes were found in samples in control Ig-Fc or untreated mice. We evaluated mBAFF levels in SCID mice with and without human MM. We have previously shown high levels of human BCMA in the plasma of MM tumor-bearing mice (Sanchez et al. Br J Haematol 2012). On days 14, 21 and 28 post-tumor implantation, mice implanted with LAGλ-1 had markedly lower plasma levels of mBAFF (P = 0.0143, P = 0.0002 and P = 0.0008, respectively) when compared to age and sex-matched mice not bearing any tumors. We obtained similar results using two additional human MM xenograft models (LAGk-1A and LAGk-2). Next, we determined whether serum BCMA levels inversely correlated with uninvolved Ig levels in MM pts. For pts with IgA (n = 134) or IgG (n = 313) MM, higher BCMA levels (〉 100 ng/ml) correlated with below normal levels of uninvolved IgG in IgA MM and uninvolved IgA in IgG MM, whereas lower BCMA levels (〈 100 ng/ml) correlated with normal uninvolved levels (P 〈 0.0001). Using the Hevylite Assay, similar results were observed for the levels of BCMA compared to uninvolved IgG isoforms in both pts with involved IgG lambda (n = 62, P = 0.0006) and IgG kappa (n = 117, P 〈 0.0001) MM. Conclusions: Our laboratory previously reported that BCMA is present in the serum of MM patients, correlates with response to treatment and predicts survival. We now demonstrate the formation of circulating BCMA-BAFF complexes in MM, and administration of recombinant BCMA to normal mice results in marked reductions in their antibody levels. We also show that BCMA levels inversely correlate with uninvolved Ig levels in MM pts. Thus, the lack of normal antibody production in MM pts results in part from circulating BCMA binding its ligands, preventing production of normal antibody-producing cells. Disclosures No relevant conflicts of interest to declare.