Subgroup Of Multiple Myeloma Patients Research Articles

Phase 2 Study of Venetoclax Plus Carfilzomib and Dexamethasone in Patients with Relapsed/Refractory Multiple Myeloma

Phase 2 Study of Venetoclax Plus Carfilzomib and Dexamethasone in Patients with Relapsed/Refractory Multiple Myeloma

Outcomes of maintenance therapy with lenalidomide or bortezomib in multiple myeloma in the setting of early autologous stem cell transplantation.

Post-transplant maintenance is widely used in multiple myeloma (MM); however, there is a lack of data on real-world outcomes. We have analyzed 577 patients with newly diagnosed MM undergoing early auto-transplantation between 2010 and 2015. A total of 341, 132 and 104 patients received no, lenalidomide (Len) or bortezomib (Bort) maintenance, respectively. Patients receiving Len or Bort maintenance had a higher incidence of high-risk cytogenetics by fluorescence in situ hybridization (31% (Len) vs 58% (Bort) vs 8% (No); P<0.001). Len maintenance led to a superior progression-free survival (PFS) compared with no maintenance (median, 37 vs 28 months, respectively; P=0.002; adjusted hazard ratio 0.48 (95% CI, 0.35-0.66)), including in subgroups with ISS stage III disease (median, 40 vs 24 months; P=0.008) and high-risk cytogenetics (median, 27 vs 16 months; P=0.032). Bort maintenance did not confer PFS benefit for the entire cohort, but improved PFS in the high-risk cytogenetic subgroup (median, 28 vs 16 months; P=0.035). Discontinuation due to toxicity was seen in 17 and 7% of patients receiving Len or Bort maintenance, respectively. Our results indicate that post-transplant maintenance with Len or Bort is well tolerated in clinical practice and improves PFS in high-risk subgroups of MM patients.

Targeting EZH2 in Multiple Myeloma Could be Promising for a Subgroup of MM Patients in Combination with IMiDs

Multiple Myeloma is a B cell neoplasia characterized by the accumulation of clonal plasma cells within the bone marrow.Epigenetics is characterized by a wide range of changes that are reversible and orchestrate gene expression. Recent studies have shown that epigenetic modifications play a role in multiple myeloma (MM) by silencing various cancer-related genes.We investigated the epigenetic genes differentially expressed between normal bone marrow plasma cells (BMPC ; N=5) and MM plasma cells from patients (N=206). Using SAM (Significance Analysis of Microarrays) analysis, only 12 genes significantly differentially expressed between BMPC and MM cells (ratio > 2 and FDR (false discovery rate) < 5%) were identified, including the EZH2 histone methyltransferase. EZH2, the enzymatic subunit of Polycomb Repressive Complex 2, is a histone methyltransferases able to repress gene expression by catalyzing H3K27me3 histone mark. EZH2 overexpression has been associated with numerous hematological malignancies, including MM. We thus studied EZH2 role in MM physiopathology and drug resistance. EZH2 expression was analyzed in normal bone marrow plasma cells (BMPCs; N=5), primary myeloma cells from newly diagnosed patients (MMCs; N=206) and human myeloma cell lines (HMCLs; N=40) using Affymetrix microarrays. EZH2 gene is significantly overexpressed in MMCs of patients (median 574, range 105 - 4562) compared to normal BMPCs (median = 432; range: 314 - 563) (P < 0.01). The expression is even higher in HMCLs (median 4481, range 581 - 8455) compared to primary MMCs or BMPCs (P < 0.001). High EZH2 expression is associated with a poor prognosis in 3 independent cohorts of newly diagnosed patients (Heidelberg-Montpellier cohort - N=206, UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N =158). Furthermore, GSEA analysis of patients with high EZH2 expression highlighted a significant enrichment of genes involved in cell cycle, downregulated in mature plasma cells vs plasmablasts, and EZH2 targets. Specific EZH2 inhibition by EPZ-6438 EZH2 inhibitor induced a significant decrease of global H3K27me3 in all the HMCLs tested (P < 0.01) and inhibited MM cell growth in 5 out of the 6 HMCLs tested. The inhibitory effect of EZH2 inhibitor on MM cell growth appeared at day 6 suggesting that it is mediated by epigenetic reprogramming. To confirm that EZH2 is also required for the survival of primary MMCs from patients, primary MM cells (n = 17 patients) co-cultured with their bone marrow microenvironment and recombinant IL-6 were treated with EPZ-6438. As identified in HMCLs, EZH2 inhibition significantly reduced the median number of viable myeloma cells by 35% (P = 0.004) from a subset of patients (n=9) while the other group (n=8) was resistant. Of interest, EPZ-6438 induced a significant global H3K27me3 decrease in both groups of patient. RNA sequencing of 6 HMCLs treated with EPZ-6438 combined with H3K27me3 ChIP analyses allowed us to create an EZ GEP-based score able to predict HMCLs and primary MM cells sensitivity to EZH2 inhibitors. We also observed a synergy between EPZ-6438 and Lenalidomide, a conventional drug used for MM treatment. More interestingly, pretreatment of myeloma cells with EPZ-6438 significantly re-sensitize drug-resistant MM cells to Lenalidomide. Investigating the effect of EPZ-6438/Lenalidomide combination in MMC, we identified that IKZF1, IRF4 and MYC protein levels were significantly more inhibited by the combination treatment (65.5%, 63.9% and 14.8% respectively) compared with Lenalidomide (51.5%, 43% and 2.2%) or EPZ-6438 (45.2%, 38.7% and 6.2%) alone.Clinical trials are ongoing with EZH2 inhibitors in lymphoma and could be promising for a subgroup of MM patients in combination with IMiDs. Furthermore, the EZ score enables identification of MM patients with an adverse prognosis and who could benefit from treatment with EZH2 inhibitors. DisclosuresGoldschmidt:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Hose:EngMab: Research Funding; Takeda: Other: Travel grant; Sanofi: Research Funding.

Smac mimetic LCL161 overcomes protective ER stress induced by obatoclax, synergistically causing cell death in multiple myeloma.

Bcl2 and IAP families are anti-apoptotic proteins deregulated in multiple myeloma (MM) cells. Pharmacological inhibition of each of these families has shown significant activity only in subgroups of MM patients. Here, we have examined a broad-spectrum Bcl2 family inhibitor Obatoclax (OBX) in combination with a Smac mimetic LCL161 in MM cell lines and patient cells. LCL161/OBX combination induced synergistic cytotoxicity and anti-proliferative effects on a broad range of human MM cell lines. The cytotoxicity was mediated through inhibition of the IAPs, activation of caspases and up regulation of the pro-apoptotic proteins Bid, Bim, Puma and Noxa by the drug combination. In addition, we observed that OBX caused ER stress and activated the Unfolded Protein Response (UPR) leading to drug resistance. LCL161, however inhibited spliced Xbp-1, a pro-survival factor. In addition, we observed that OBX increased GRP78 localization to the cell surface, which then induced PI3K dependent Akt activation and resistance to cell death. LCL161 was able to block OBX induced Akt activation contributing to synergistic cell death. Our results support clinical evaluation of this combination strategy in relapsed refractory MM patients.

TP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) stable silencing increases late apoptosis by upregulation of caspase 9 and APAF1 in RPMI8226 multiple myeloma cell line

BackgroundTP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) modulates apoptotic pathways preventing the formation of the apoptosome complex. Our group previous study showed that 90% of patients' multiple myeloma (MM) marrow-derived plasma cells present TRIAP1 overexpression as compared to normal plasma cells. Due to high prevalence and lack of information on TRIAP1's role in MM biology, we decided to explore the impact of TRAIP1 through stable gene silencing in MM cell lines and its effect on cell cycle and apoptosis. MethodsTRIAP1 expression was examined in MM cell lines by quantitative real time PCR. Cell lines were submitted to transduction with lentiviral vector encoding a TRIAP1-specific short hairpin RNA (shRNA) and, as control, encoding a non-targeting shRNA (scramble). Apoptosis was assessed by flow cytometry with annexin V and propidium iodide staining (the later also used for cell cycle), APAF1 and Caspase 9 apoptosome related genes expression and Caspase 9 and Caspase 3/7 activity. ResultsRPMI8226 and U266 cell lines were chosen for transduction experiments since they present higher levels of TRIAP1 expression. Inhibition of TRIAP1 in RPMI8226 cells increased the percentage of apoptotic cells, accompanied by increased expression of APAF1 and Caspase 9, and Caspase 9 and Caspase 3/7 activity. Transduced U266 cell line did not show sustained inhibition of TRIAP1 expression nor apoptosis induction. ConclusionStable silencing of TRIAP1 induces late apoptosis through APAF1/Caspase 9 pathway at least in RPMI8226 cell line, suggesting that it could be exploited as a potential target at least for a subgroup of MM patients. General significanceIn the present study, we demonstrated effects of TRIAP1 silencing on RPMI8226 MM cell line and established its mechanism mediated through APAF1 and Caspase 9. No relevant effect was found after gene silencing in U266 cell line.

Maternal Embryonic Leucine Zipper Kinase (MELK): a Novel Marker of Poor Prognosis and Attractive Drug Target in High-Risk Patients with Multiple Myeloma

Background:Despite significant progress in the treatment of multiple myeloma (MM) through the introduction of immunomodulatory drugs and proteasome inhibitors, therapeutic progress is limited for high-risk patients. Therefore, it is essential to define and validate novel drug targets in myeloma to implement personalized treatment options, predict drug activity and finally improve treatment outcome for all subgroups of MM patients. In the current study we aimed to analyse the prognostic value of maternal embryonic leucine zipper kinase (MELK) in MM and investigated the activity of a small molecule inhibitor of MELK (OTSSP167).Methods:MELK expression levels were analysed in two large cohorts of publically available gene expression (GEP) datasets (GSE2658 and GSE9782; n=551 and n=264, respectively) and 8 human myeloma cell lines (HMCLs). The utility of MELK as potential drug target in MM was investigated by using a selective small molecule inhibitor against MELK (OTSSP167). HMCLs were treated at varying concentrations (0-1000 nM) and analysed for viability, apoptosis and cell cycle status. Regulatory networks involved in the mechanisms of OTSSP167 were revealed by quantitative PCR (qPCR) and proteomic profiling of HMCLs after short-term (5 hours) treatment with OTSSP167 at 25-50 nM.Results:The prognostic impact of MELK was studied in two publically available GEP-datasets (GSE2658 and GSE9782). Interestingly, MELK expression was significantly elevated in the GEP-defined high-risk proliferation associated molecular subgroup (P<0.001). High levels of MELK expression were accordingly associated with poor prognosis. We observed significantly reduced overall survival in patients with high compared to low MELK levels treated within the total therapy 2 (P =0.0003), total therapy 3 (P =0.04) and the APEX trial protocol (P =0.002).These results pointed to a role for MELK as potential drug target in high-risk patients. To test this, we used a highly selective inhibitor of MELK (OTSSP167). In line with their proliferative character, MELK expression was detected in 8 of 8 HMCLs. Treatment with OTSSP167 led to a dose-dependent reduction of viability in all HMCLs tested (median IC50: 10.16 nM, range: 7.6 - 15.2 nM). Importantly, we also detected synergistic and additive drug activity of OTSSP167 in combination with pomalidomide and carfilzomib.OTSSP167 induced apoptosis in all HMCLs investigated, verified by annexin V/7-AAD staining, detection of cleaved PARP and mitochondrial membrane depolarization. The apoptotic effects might be attributed to a significant downregulation of IRF4 (up to 0.75±0.27 fold reduction, P =0.009) and MCL-1 (up to 0.51±0.09 fold reduction, P =0.002) expression. We also observed accumulation of MM cells in the G2 phase of the cell cycle (OPM-2: 24% vs 42%, MM.1S: 23% vs 38.8% of cells in G2 phase without or with OTSSP167 at 25 nM for 48 hours, P<0.001). This was associated with downregulation of central genes involved in the propagation of the cell cycle, including PLK-1, cyclin B1 and aurora kinase A (up to 0.82±0.08, 0.93±0.04 and 0.88±0.08 fold reduction, respectively; P<0.0001). We also detected downregulation of cyclin D1 (up to 0.41±0.3 fold reduction, P =0.03). These results were confirmed at the protein level by using proteomic profiling of three HMCLs after 5 hours of treatment with OTSSP167 at 50 nM. Again, we observed deregulation of proteins involved in the initiation of mitosis (cyclin B1, aurora kinase A, nucleolin). Of note, we also detected upregulation of several proteins involved in protein folding and stabilization (e.g. Csp, HSP90 alpha and HSP90 beta) as well as glycolysis (e.g. ENO1, ALDOA, G3P2, TPI1), suggesting that these factors might regulate potential drug resistance mechanisms.Conclusion:Our findings reveal MELK expression as a novel poor prognostic marker in newly diagnosed and relapsed MM, depicting a group of high-risk patients. Inhibition of MELK with OTSSP167 led to the induction of apoptosis and cell cycle arrest by targeting central genes of the MM signaling network (e.g. MCL-1, cyclin D1, aurora kinase A). Moreover, combination of OTSSP167 with established anti-MM drugs showed synergistic activity. These results emphasize to initiate further pre-clinical and clinical testing of MELK inhibition as a novel drug target in MM, especially for patients at high-risk. DisclosuresLudwig:Takeda: Research Funding; Celgene Corporation: Honoraria, Speakers Bureau; Onyx: Honoraria, Speakers Bureau; Bristol Myers Squibb: Honoraria, Speakers Bureau; Janssen Cilag: Honoraria, Speakers Bureau.

Overcoming Resistance to Apoptosis in Multiple Myeloma By Simultaneous Inhibition of Bcl2 and IAP Families of Anti-Apoptotic Proteins

Background: Multiple myeloma (MM) cells evade apoptosis through multiple mechanisms thus enabling it to evade therapy. The Bcl2 family of anti-apoptotic proteins is aberrantly expressed in MM cell lines and patient cells. Yet, pharmacological intervention of this family appears to have significant activity only in molecular subgroups of MM patients. This clearly suggests alternate mechanisms of overcoming apoptotic signals in MM cells in addition to the Bcl2 family, through proteins such as IAPs. We have previously shown that simultaneous inhibition of the three major IAP proteins, namely cIAP1, cIAP2 and XIAP is required to induce pronounced apoptosis in MM cells. However, IAP inhibition results in apoptosis in only some MM cell lines and patient cells. Given that levels of Bcl2 family proteins are unaffected by IAP inhibition, we hypothesized that combined inhibition of the IAP proteins using a SMAC mimetic LCL161 and the Bcl2 family proteins using a pan-Bcl2 inhibitor obatoclax (OBX) will lead to more pronounced and synergistic cell death in a broader subgroup of MM patients.Methods: LCL161 was synthesized by Novartis Inc. (Basel, Switzerland). OBX was purchased from Selleckchem (Houston, USA). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and patient cells. Immunoblotting was done on cell extracts at various time points following incubation with the drugs in order to study the cell signaling pathways and aResults: LCL161/OBX combination induced synergistic cytotoxicity and anti-proliferative effects on a broad range of human MM cell lines, including drug resistant cell lines like DOX40 and MM1R. Components of the bone marrow microenvironment including bone marrow stromal cells and tumor promoting cytokines (VEGF, IGF and IL6) were unable to protect MM cells from the effects of the drug combination. We saw a time dependent increase in apoptosis, with the combination inducing significantly more apoptosis than either of the single agents alone. Examining the mechanism of action of the drug combination showed clear inhibition of the IAP proteins, activation of caspases 9, 8, 3 and Bid by LCL161 and the combination and up regulation of the pro-apoptotic proteins Bim, Bid, Puma and Noxa and accumulation of LC3-II by OBX and the combination. Using chloroquine along with the OBX, we were able to demonstrate that OBX induced protective autophagy and the addition of LCL161 was able to overcome this protective effect induced after single agent OBX treatment. Since protective autophagy can be induced by the ER stress response, we then examined the expression levels of proteins involved in this pathway. We observed clear induction of ER stress mediated UPR pathway by both the drugs. However, LCL161 and OBX induced different branches of the UPR pathway. OBX activated the ATF6 and pErk/peif2α/ATF4 branches of the UPR, both of which have been implicated in cell survival during ER stress. ATF4 under irrecoverable ER stress can lead to increase in transcription of CHOP and cause apoptosis. We therefore examined levels of CHOP and observed no induction of CHOP post treatment with either of the drugs or the combination. LCL161, however differentially modulated the IRE1 branch of the UPR by down regulating Xbp-1 splicing, which is a pro survival activity of IREI and up regulating pJNK, which indicated a pro-apoptotic activity induced by IRE1 post irrecoverable ER stress This indicated that the ER stress induced apoptosis is triggered by LCL161, which might be important to overcome the ER induced protective effects induced by OBX.Conclusion: Taken together, our studies indicate that LCL161/OBX combination induces synergistic cell death through modulation of apoptosis, authophagy and the ER stress response. DisclosuresNo relevant conflicts of interest to declare.

Weighted Gene Co-Expression Network Analysis (WGCNA) Identifies Highly Proliferative Myeloma Subgroup Responsive to CDK4/ARK5 Inhibition

Multiple myeloma (MM) is an incurable plasma cell malignancy accounting for more than 10,000 deaths in the US each year. Hence the pursuit for novel therapeutic agents remains critically important. Myeloma pathogenesis is associated in part with aberrant cell cycle progression. Inhibition of cyclin dependent kinases CDK4/6 results in cell cycle arrest and sensitization to Bortezomib and other active agents in MM (Huang, Blood 2012). Here, we show that ARK5, a novel member of the human AMPKfamily, is overexpressed in 70% of MM and helps promote proliferation and cell cycle progression via G1/S phase activation in an mTOR dependent manner.We examined the role of ARK5 using loss of function studies by ARK5 siRNA transfection in MM1.S, NCI-H929 cells as well as treatment with ON 123300, a dual CDK4/ARK5 kinase inhibitor. ARK5 siRNA knockdown decreased MM cell viability and cell proliferation via G1/S arrest compared to control siRNA. ARK5 siRNA treatment significantly (~70%) induced apoptosis in MM cells as detected by Annexin V/PI staining. We observed that phosphorylation of Rb, a critical cell cycle protein was significantly reduced in ARK5 depleted cells. Moreover, mTOR pathway inhibition was confirmed by reduction of pS6K in ARK5depleted cells as compared to control siRNA treated cells.ON 123300 decreased viability in MM cell lines and patient cells but was not lethal to normal PBMCs. A single treatment of 50nM drug stratified MM cell lines into 2 groups, 5 resistant (MM.1R, KMS11, U266, RPMI-8226 and ARP1) and 4 sensitive cell lines (>80% cell kill-MM.1S, EJM, JJN3, NCI-H929). ARK5 protein expression by western blot analysis was much higher in sensitive cell lines. ON 123300 triggered G0/G1 cell cycle arrest and induced apoptosis similar to the effect of ARK5 siRNA (80% vs 70%). ON 123300 treatment also reduced phosphorylation of pRb and pS6K downstream of mTOR pathway. These results confirm that cell inhibitory effects of ON 123300 in MM are mediated in a large part via inhibition of ARK5. Co-culture experiments with BMSCs showed that ON 123300 not only targets MM cells but also overcomes the cytoprotective effects of the MM-host BM microenvironment. 4/5 ARK5 positive primary samples with adverse cytogenetics including 1q amplification and CyclinD1 translocation were sensitive to ON 123300 (>80% cytotoxicity) at 50nM. Further, IP injection of ON 123300 (100mg/kg) in tumor xenograft models (MM1.S, NCI-H929) showed that ON 123300 is well tolerated and significantly inhibits tumor growth in vivo(p<0.001).To study the mechanism of action for ON 123300, we performed geneset enrichment analysis (GSEA) on drug induced gene expression signature of RNA-Seq data from pre-post treated cell lines. We interrogated a wide array of geneset libraries, including MSigDB (Subramanian, PNAS 2005), drug induced transcriptional modules (Iskar, Mol Sys Bio. 2013) and disease signatures (Sirota, Sci Transl Med 2011). GSEA showed significant representation of genes that are enriched in normal plasma cells and rapamycin sensitive geneset.Next, we developed a weighted co-expression network (WGCNA, Langfelder BMC Bioinformatics 2008) based classifier using 304 MM samples from MMRC collection and RNA-Seq from 28 MM patients from Mt. Sinai Hospital. WGCNA defines a network that continuously links all genes and then clusters the most highly co-expressed genes in defined modules. These network modules were associated with clinical traits and UAMS GEP classification (Zhan, Blood 2006) of each sample. There was significant overlap between highly proliferative “PR” and “Cyclin D1/2” patient subsets based on classification and sensitivity to CDK4/ARK5 inhibition. Our classifier accurately discriminated 4 sensitive primary samples from one resistant sample, all tested in vitro. All sensitive samples were either Cyclin D1/2 or PR as per UAMS classification.Our preclinical studies provide the basis for clinical evaluation of CDK4/ARK5 inhibitor ON 123300 due to its selective cytotoxicity on MM cells in vitro and in vivo. Using WGCNA we establish a systematic framework by constructing for the first time, MM-associated gene co-expression networks contributing to tumorigenesis and progression. Thus, WGCNA modeling is a novel approach for identification of MM patient subgroups that have a higher likelihood of response in clinical trials with CDK4/ARK5 inhibitors. DisclosuresChari:Array BioPharma: Scientific Advisory Board Other; Celgene Corporation: Scientific Advisory Board, Scientific Advisory Board Other; Millennium Pharmaceuticals, Inc: Scientific Advisory Board, Scientific Advisory Board Other; Onyx Pharmaceuticals, Inc.: Scientific Advisory Board, Scientific Advisory Board Other. Reddy:Onconova Therapeutics, Inc.: Consultancy, Patents & Royalties. Reddy:Onconova Therapeutics, Inc.: Consultancy, Patents & Royalties. Dudley:Ecoeos, Inc: Consultancy, Equity Ownership; GNS Healthcare: Consultancy; GlaxoSmithKline: Consultancy; Ayasdi, Inc: Equity Ownership; NuMedii, Inc: Equity Ownership; Ubalo, Inc.: Equity Ownership. Jagannath:Onyx Pharmaceuticals, Inc.: Consultancy; Celgene Corporation: Scientific Advisory Board, Scientific Advisory Board Other; Millennium Pharmaceuticals, Inc. : Scientific Advisory Board, Scientific Advisory Board Other.

Combination of international scoring system 3, high lactate dehydrogenase, and t(4;14) and/or del(17p) identifies patients with multiple myeloma (MM) treated with front-line autologous stem-cell transplantation at high risk of early MM progression-related death.

To construct and validate among patients with multiple myeloma (MM) who were treated with intensive therapy a prognostic index of early MM progression-related death. Patient-level data from the Intergroupe Francophone du Myélome (IFM) 2005-01 trial (N = 482) were used to construct the prognostic index. The event was MM progression-related death within 2 years from treatment initiation. The index was validated using data from three other trials: the Gruppo Italiano Malattie Ematologiche dell' Adulto (GIMEMA) 26866138-MMY-3006 trial (N = 480), the Programa para el Estudio de la Terapéutica en Hemopatía Maligna (PETHEMA)-GEMMENOS65 trial (N = 390), and the Hemato-Oncologie voor Volwassenen Nederland (HOVON) -65/German-Speaking Myeloma Multicenter Group (GMMG) -HD4 trial (N = 827). The risk of early MM progression-related death was related to three independent prognostic variables: lactate dehydrogenase (LDH) higher than than normal, International Staging System 3 (ISS3), and adverse cytogenetics [t(4;14) and/or del(17p)]. These three variables enabled the definition of an ordinal prognostic classification composed of four scores (0 to 3). Patients with a score of 3, defined by the presence of t(4;14) and/or del(17p) in addition to ISS3 and/or high LDH, comprised 5% (20 of 387 patients) to 8% (94 of 1,139 patients) of the patients in the learning and validation samples, respectively, and they had a very poor prognosis. When applied to the population of 855 patients who had received bortezomib-based induction therapy in the four trials, the prognostic classification was also able to segregate patients into four categories, with a very poor prognosis attributed to patients with a score of 3. Our model allows the simple definition of a subgroup of MM patients at high risk of early MM progression-related death despite the use of the most modern and effective strategies.

Polyclonal serum IgM level identifies a subgroup of multiple myeloma patients with low-risk clinicobiological features and superior survival.

Normal plasma cells (PCs) are either undetectable or outnumbered by the myelomatous PC compartment in bone marrow of multiple myeloma (MM). However, residual normal PCs have been detected in a minority of symptomatic MM patients with superior survival. The number of normal PCs is also an important factor to identify monoclonal gammopathy of undetermined significance (MGUS)-like MM. We speculate that the polyclonal serum IgM level in non-IgM myelomas may reflect the number of residual normal PCs. Here we investigated the prognostic relevance of polyclonal serum IgM level in a series of 485 newly diagnosed symptomatic MM (NDMM) patients. Our results showed that symptomatic MM patients with polyclonal IgM more than 0.5g/L displayed a favorable baseline clinical feature, together with a significantly lower frequency of high-risk cytogenetic abnormalities. This group of patients had a significantly prolonged progression-free survival (PFS) and overall survival (OS) regardless of thalidomide or bortezomib therapy. Furthermore, the superior outcome was independent of the depth of response. Our findings suggest that polyclonal IgM level is capable of identifying a group of symptomatic MM patients with distinct clinicobiological characteristics and favorable survival, similar with MGUS-like MM.

Combined targeting of MEK/MAPK and PI3K/Akt signalling in multiple myeloma

So-called RAS-dependent pathways, such as those signalling via mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3K)/Akt, are implicated in proliferation and survival of multiple myeloma (MM) cells. However, the effects of their combined blockade and its potential therapeutic utility for the treatment of RAS-mutated MM have not systematically been analysed. Here, we tested the functional consequences of single versus combined inhibition of the MEK/MAPK and PI3K/Akt pathways in a large series of primary MM samples (n = 55) and MM cell lines (n = 11). Additionally, the anti-myeloma activity of different treatments was analysed with respect to the RAS mutation status. PI3K/Akt blockade was generally more pro-apoptotic than blockade of MEK/MAPK both in cell lines and in primary MM samples. Simultaneous blockade of both pathways led to significantly enhanced anti-myeloma activity in 75% of primary MM samples, whereas the remainder was largely resistant. Resistance to combination blockade was exclusively observed in RAS wildtype cases, whereas sensitivity was noted in RAS wildtype and in RAS mutated MM. These results suggest that oncogenic RAS is a predictor of sensitivity to combination treatment with PI3K/Akt and MEK/MAPK inhibitors and that such an approach might therefore be beneficial for this genetically well-defined subgroup of MM patients.

Abstract 1873: Gene expression analysis of newly diagnosed Multiple Myeloma (MM) patients carrying amplified MDM4 and/or deleted p53

Abstract The p53 tumor suppressor pathway is tightly kept in check or completely silenced in cancer cells. A potent inhibitor of p53 is MDM4, which is located on chr.1q: it is critical for the control of p53 activity during the response to stress and is often amplified in several types of cancer. Since both del(17p) and amp(1q) identify a subgroup of high-risk MM pts, even when the novel agents are part of up-front treatment strategy, we molecularly analyzed a subgroup of MM patients treated with bortezomib-thalidomide-dexamethasone (VTD) and autologous stem cell transplantation (ASCT) in order to investigate mechanisms which might be activated in myeloma plasma cells to direct and/or indirect limit the p53 function. CD138+ plasma cells obtained at diagnosis from 61 pts, subsequently treated with VTD and ASCT were analysed by means of GEP and unpaired analysis of CNA (Affymetrix U133 Plus2.0 and 6.0 SNP arrays). Nineteen out of 61 pts (31%) carried a minimal amplification region on chr.1q, harboring MDM4. Eight out of 61 pts (13%) carried a 482 Kb minimal deletion region on chr.17 harboring TP53. To explore the involvement of the p53 pathway in MM, pts were stratified according to the presence of amplified MDM4 and/or deleted p53 (group A, 24 pts) or the absence of both these abnormalities (group B, 37 pts). Baseline clinical characteristics were homogeneous, except for a higher rate of ≤ 3 bone lesions in pts of group A. The rate of best complete or near complete response was 40% in group A and 14% in group B (p = 0.04). With a median follow-up of 36 months, the risk of progression was 46% for pts in group A and 19% for those in group B (p = 0.03). The average number of aberrations per group was overall higher in group A as compared to group B (165 vs. 103 CNAs, p =0.03). A comparison of expression profiles of the two subgroups of pts highlighted a deregulated expression of genes involved in the mechanisms of response to genotoxic stress, i.e. of damage sensor genes (ATM, RAD9, RAD 50, ATRip), of damage signal mediator genes (H2AFX, 14-3-3), of genes involved in regulation of cell proliferation (CDK6, CDC25, CCND2) and of anti-apoptotic genes (BCL2, p73) (one-way ANOVA plus multiple-test correction with FDR &amp;lt;0,05). Finally, this group of pts significantly over-express the transcription factor YY1, which is known to interact with p53, thus inhibiting its transcriptional activity. In conclusion, pts carrying amplified MDM4 and/or deleted p53 showed a significantly higher number of CNAs and the significant over-expression of genes involved in the response mechanisms to genotoxic stress, as compared to pts lacking these chromosomal aberrations. This might account for the worse outcome of patients harboring del(17p) and/or amp(1q). The MDM4 locus amplification and the YY1 over-expression might contribute to maintain p53 in an OFF state by an indirect mechanism. Supported by: Fondazione Del Monte di Bo e Ra, Ateneo RFO grants (M.C.) BolognAIL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1873. doi:1538-7445.AM2012-1873

Bisphosphonate Anticancer Activity in Multiple Myeloma

Bisphosphonates inhibit osteoclast-mediated bone resorption and have been used extensively to prevent skeletal-related events in patients with bone lesions from multiple myeloma (MM). In addition, in vitro and in vivo preclinical data suggest that bisphosphonates also have antimyeloma properties that may induce myeloma cell apoptosis, activate an anticancer immune response, inhibit angiogenesis, and reduce tumor burden, supporting an expanded role for bisphosphonates. Signals for improved survival in the clinic first emerged in retrospective analyses of MM patient subgroups in larger clinical trials. Recently, improved progression-free survival and overall survival with bisphosphonates have been reported in the overall populations of large-scale randomized clinical trials. Several ongoing clinical trials will help further define the role of bisphosphonates during antimyeloma therapy. Overall, bisphosphonates appear to be well tolerated in patients with MM; the most common adverse events are mild and can be easily managed. However, emphasis on renal monitoring and preventive dentistry are necessary to reduce the risk of potential adverse events, and have become the standard of care for patients with MM.

High Number of Copy Number Alterations and Over-Expression of Genes Involved in the Response Mechanisms to Genotoxic Stress Both Characterize Newly Diagnosed Multiple Myeloma (MM) Patients Carrying Amplified MDM4 and/or Deleted p53,

Abstract 3935 BackgroundThe p53 tumor suppressor pathway is tightly kept in check, or completely silenced in cancer cells. A potent inhibitor of p53 is represented by MDM4, which is critical for the control of p53 activity during the response to stress and is often amplified in several types of cancer. TP53 mutations are rare in newly diagnosed MM, while occur more frequently as late event in the course of the disease and are related to survival. Recently, the adverse prognostic impact of chr. 1q amplification, described in almost 40% of newly diagnosed MM pts, has been reported. The minimal amplified region on chr. 1q harbors MDM4. Since both del(17p) and amp(1q) identify a subgroup of high-risk MM pts, even when the novel agents are part of up-front treatment strategy, we molecularly analyzed a subgroup of MM patients treated with bortezomib-thalidomide-dexamethasone (VTD) incorporated into autologous stem cell transplantation, in order to investigate mechanisms which might be activated in myeloma plasma cells to direct and/or indirect limit the p53 function. MethodsThirty eight pts treated with VTD incorporated into autologous stem cell transplantation were analysed by means of gene expression profile (Affymetrix U133 Plus2.0 array) and unpaired analysis of copy number alterations (CNA) (Affymetrix 6.0 SNP array). Both GEP and SNP arrays experiments were performed on highly purified CD138+ bone marrow plasma cells obtained at diagnosis from each pts. The presence of CNAs in chr.1 and 17 was evaluated to identify pts carrying amp(1q) and del(17p). ResultsEighteen out of 38 pts (42%) carried a minimal amplification region of 1,1 Mb on chr.1q, which harbors MDM4. Five out of 38 pts (13%) carried a minimal deletion region of 482 Kb on chr.17, which harbors TP53. To explore the involvement of the p53 pathway in MM, pts were stratified according to the presence of amplified MDM4 and/or deleted p53 (group A, 18 pts) or the absence of both these abnormalities (group B, 20 pts). Baseline clinical characteristics were homogeneous, except for a higher rate of ≥ 3 bone lesions in pts carrying amplified MDM4 and/or deleted p53. The rate of best complete or near complete response was 89% in group A and 75% in group B. With a median follow-up of 36 months, the risk of relapse or progression was 50% for pts in group A and 25% for those in group B. The average number of aberrations per group was overall higher in group A as compared to group B (165 vs. 103 CNAs, p =0.03); indeed, the presence of amplified MDM4 and/or deleted p53 was significantly associated with a list of 95 CNAs (clustered on chr. 1, 2, 6, 8, 11, 13, 16 and 18), which included del16q (with a minimal area of deletion including WWOX), observed in 39% vs. 5% cases from group A and B, respectively (p<0.05) and chr.8 aberrations (with amplifications and or deletions, both including TRPS1) observed in 61% vs. 15% cases from group A and B, respectively (p<0.05). A comparison of expression profiles of patients carrying or not amplified MDM4 and/or deleted p53 confirmed the over-expression of MDM4 in the former group of pts and highlighted in these pts an overall activation of genes involved in the response mechanisms to genotoxic stress: indeed a significant over-expression of damage sensor genes (ATM, RAD9, RAD 50, ATRip), of damage signal mediator genes (H2AFX, 14-3-3), of genes involved in regulation of cell proliferation (CDK6, CDC25, CCND2) and of anti-apoptotic genes (BCL2, p73) was observed in pts with amplified MDM4 and/or deleted p53 (one-way ANOVA plus multiple-test correction with FDR <0,05). Finally, this group of pts significantly over-express the transcription factor YY1, which is known to interact with p53, thus inhibiting its transcriptional activity. ConclusionsPts carrying amplified MDM4 and/or deleted p53 showed a significantly higher number of CNAs and the significant over-expression of genes involved in the response mechanisms to genotoxic stress, as compared to pts lacking these chromosomal aberrations. This might account for the worse outcome of patients harboring del(17p) and/or amp(1q). The amplification of MDM4 locus and the over-expression of YY1 might contribute to maintain p53 in an OFF state by an indirect mechanism. Additional data on the role of both direct and indirect control of p53 pathway on VTD-treated MM pts prognosis, extended to an higher number of pts, will be presented during the meeting.Supported by: Fondazione Del Monte di Bo e Ra, Ateneo RFO grants (M.C.) BolognAIL. Disclosures:Cavo:Genzyme: Honoraria.

Biological and Clinical Significance of CD81 Expression by Clonal Plasma Cells in High-Risk Smoldering and Symptomatic Multiple Myeloma (MM) Patients,

Abstract 3936The presence of CD19 in myelomatous plasma cells (MM-PC) correlates with adverse prognosis in MM. Although CD19 expression is up-regulated by CD81, this marker has been poorly investigated and its prognostic value in myeloma remains unknown.Herein, we assessed the frequency and the prognostic value of the immunophenotypic detection of CD81 surface expression in MM-PC of high-risk smoldering (SMM) and symptomatic MM patients at diagnosis and its role as a potential therapeutic target. The study included 230 elderly MM patients treated according to the Spanish GEM05>65y trial, and a validation set based on 325 transplant candidates MM patients enrolled in the GEM05<65y trial was used. In addition we analyzed a total of 56 high-risk SMM patients. The median follow-up was 32 and 22 months for the MM and SMM, respectively. Expression of CD81 on MM-PC was assessed by multiparameter flow cytometry (MFC). FISH was performed at baseline in immunomagnetic-enriched PCs from 211 of the 230 elderly MM patients, and in a subset of these patients (N=23) mRNA gene expression profiling (GEP) was also performed.MFC immunophenotyping showed the presence of CD81+ MM-PC in 20 of 56 (36%) SMM, and 90 of 230 (39%) MM patients. CD81+ SMM cases had a shorter TTP to symptomatic disease than CD81− patients (NR vs 37 months, P =.02). Similarly, CD81+symptomatic MM patients showed shorter PFS (3y: 29% vs 48%, P <.001) and OS (3y: 64% vs 76%, P =.008) rates compared to CD81− cases. Multivariate analysis including other baseline variables showed that the best combination of independent predictive parameters for PFS were: CD81+ expression on MM-PC (HR=1.8; P =.004), high-risk cytogenetics (HR=1.8; P =.02) and percentage of MM-PC in S-phase (>2%; HR=1.7; P =.02); in turn for OS, CD81+ expression (HR=2.2; P =.005), high-risk cytogenetics (HR=2.2; P =.006) and age (≥75 years; HR=1.8; P =.03) were selected. To validate the adverse impact of CD81+ expression, we explored whether this new marker would retain its prognostic value in a series of 325 transplant candidates, symptomatic MM patients. CD81+ cases (138 out of 325, 42%) showed shorter PFS (3y: 44% vs 62%, P <.001) and OS (3y: 74% vs 89%, P =.004) rates as compared to CD81− cases.The prognostic influence of CD81 expression prompted us to investigate its potential role as a therapeutic target in MM cell lines. 5 out of the 13 cell lines analyzed were homogeneously positive for CD81 (RPMI-8226, RPMI-LR5, NCI-H929, OPM-2, JJN3) while the others exhibited no expression; these results were further validated by western blotting. We then tested the effect of two different anti-CD81 antibodies on cell proliferation on 2 CD81+ cell lines (RPMI-8226 and JJN3) as well as in 1 CD81− (MM1S) MM cell line. Interestingly, the two antibodies tested decreased cell proliferation (around 30%, P ≤.005) in the two CD81+ cell lines, whereas no differences were found for the CD81− MM1S cell line. To assess whether anti-CD81 antibodies facilitate immune effector cell function, we performed in vitro ADCC. However, no effect was noted in the CD81+ RPMI-8226 cell line, using either PBMCs or the macrophage cell line RAW264.7, and at different cell ratios. Similar results were also found upon using CDC assay in CD81+ RPMI-8226 and JJN3 cell lines.Finally, we explored in a subgroup of MM patients (n=23) in which information was available, the relationship between positivity for CD81 by MFC and its genomic expression. The expression level of CD81 mRNA was significantly decreased in CD81− MM cases (P <.001) vs. both healthy adults and CD81+ patients (P <.001), with no significant differences between these two latter subgroups. Moreover, we found a highly significant correlation between the expression of CD81 mRNA by GEP and the percentage of CD81+ MM-PC by MFC (r=.812; P <.001). We further investigated by GEP, differences in other genes involved in the CD81 signaling pathway, and CD81+MM patients showed significantly higher levels of CD79A (P =.03) and SYK (P =.02) mRNA than CD81−cases.In summary, our findings show the existence of a phenotypic-genomic correlation of CD81 expression in patients with myeloma. Expression of CD81 in MM-PC is an independent prognostic factor for patients with symptomatic MM and a marker for risk of progression in SMM. Blockage of CD81 with anti-CD81 antibodies does not show significant anti-myeloma activity; therefore, the precise mechanism of CD81 activation of MM-PC deserves further investigations. Disclosures:Paiva:Celgene: Honoraria; Janssen: Honoraria. Rosiñol:Janssen: Honoraria; Celgene: Honoraria. Mateos:Janssen: Honoraria; Celgene: Honoraria. Lahuerta:Janssen: Honoraria; Celgene: Honoraria. Blade:Janssen: Honoraria; Celgene: Honoraria. San Miguel:Janssen-Cilag: Honoraria; Celgene: Honoraria.

Defining the biological subgroup of multiple myeloma patients which benefits maximally from the overall survival (OS) benefit associated with treatment with zoledronic acid (ZOL).

8083 Background: Myeloma IX examined thalidomide-based induction and maintenance in patients (pts) with multiple myeloma (MM) treated in intensive or non-intensive pathways and SRE reduction with bisphosphonates (BPs; IV ZOL vs oral clodronate [CLO]). In 1960 evaluable pts, ZOL improved OS by 5.5 mo (P = .04) and significantly reduced SRE risk vs CLO (Morgan et al. Lancet. 2010). A biologic basis for this benefit has not been reported. Methods: Here we discuss subgroup analyses (demographics and biologic disease characteristics) that can inform our understanding of how ZOL OS benefit is derived. Baseline bone disease (BD+ or BD−) was screened using axial skeletal surveys. FISH analysis of CD138-selected plasma cells was performed using standard methodology on a subset (n = ~1000); gene expression was assessed using the Affymatrix system. Results: In subgroup analyses, ZOL OS benefit vs CLO was not affected by gender, ISS stage, or specific myeloma genotypes. BD rates at presentation varied between genotypes (higher with hyperdiploidy and t[11:14], lower with t[4;14]). Integration of FISH and ISS data to better delineate high- and low-risk myeloma biology was performed, and evaluation of effects on ZOL’s OS benefits is underway. Previously, among BD− pts (~30% of total), ZOL significantly decreased SRE risk (~30%) vs CLO. For the first time, we report that OS was significantly shorter in BD+ (median 45.5 mo, n = 1401) vs BD− pts (median 51.6 mo, n = 540; P =.009). OS benefit with ZOL vs CLO was apparently most profound in BD+ pts, with no significant OS difference between BPs in BD− pts. Differences in gene expression profiles and myeloma-bone interactions in BD+ and BD– pts may explain the varying OS benefit despite similar SRE prevention with ZOL. Conclusions: ZOL OS benefits vs CLO are independent of demographic and baseline disease characteristics evaluated except for BD. ZOL more effectively reduced SREs than CLO in both BD+ and BD− pts, but OS benefits were restricted to BD+ pts. Further investigations of gene expression profiles are ongoing and may provide further insight into differential interactions between these BPs and myeloma in bone marrow.

Genetic polymorphisms associated with outcome in multiple myeloma patients receiving high-dose melphalan

High-dose melphalan (HDM) is an essential component in the treatment of patients with multiple myeloma (MM). Few data are available regarding genetic polymorphisms associated with patient outcome or toxicity in this setting. To identify such polymorphisms, we performed a retrospective analysis, genotyping single nucleotide polymorphisms (SNPs) with the arrayed primer extension (APEX) technology in 169 patients having received HDM for MM. We analyzed 209 SNPs in 95 genes involved in drug metabolism, DNA repair, cell cycle and apoptosis. SNPs in ABCB1, CYP3A4 and TP53BP2 were associated with response to VAD induction therapy (P<0.01). SNPs in ALDH2, GSTT2 and BRCA1 were associated with response to HDM (P<0.01). Polymorphisms in CYP1A1, RAD51 and PARP were associated with disease progression whereas polymorphisms in ALDH2 and CYP1A1 were correlated with OS. Polymorphisms in BRCA1, CDKN1A and XRCC1 were associated with the occurrence of severe mucositis after HDM. These results suggest that SNPs of genes involved in drug metabolism or DNA repair could be used to distinguish MM patient subgroups with different toxicity/efficacy profiles.

MicroRNAs Are Differentially Expressed Among Cytogenetic Subgroups in Multiple Myeloma.

Abstract 2833Poster Board II-809Micro-RNAs (miRNAs) are a class of small non-coding single stranded RNAs of 20-22 nucleotides in length. MiRNAs regulate gene expression by binding to the 3' UTR of target mRNAs, which leads to either mRNA degradation or translational inhibition of this target mRNA. MiRNAs play a critical role in cell growth, survival and differentiation and have been suggested to function as tumor suppressor and oncogenes, and/or play a critical role in the pathogenesis of Multiple Myeloma (MM). We investigated miRNA expression in 41 newly diagnosed patients with MM who were included in a clinical trial. MiRNA expression profiling was performed in BM derived CD138 selected plasma cells (PC) obtained from newly diagnosed MM patients with a minimum monoclonal PC purity of > 80%. A quantitative PCR-method (TaqMan low density arrays-microRNA assay', Applied Biosystems) was used for miRNA quantification. The relative expression levels of miRNAs were calculated using the 2-DDCt method and the data was normalized using the endogenous control RNU48. 176/365 miRNAs (48.2%) were expressed in these MM patients. Unsupervised hierarchical clustering based on the average-linkage method and principal component analysis (PCA) was performed in Partek software (Partek Genomics Suite), showing a differential hierarchy for the chromosomal translocation or gain subgroups. When patients with or without chromosome 13 deletions were compared, no correlation was found between expression levels of miRNAs located on chromosome 13 and the deletion status of this chromosome as determined by FISH. Three miRNAs, i.e. miRNA-126, miRNA-145 and miR-517c were identified that were different between MM patients with or without chromosome 13 deletion. These miRNAs were not located on chromosome 13. Likewise, when comparing the miRNA expression in MM patients with or without chromosome 1q gain, the three most differentially expressed miRNAs were miRNA-23a, miRNA-200a# and miRNA-145, which are not on chromosome 1. In addition, miR-18a, located on chromosome 1 was upregulated in MM patients with 1q gain. MM patients with t(4;14) showed differential expression of miRNA-520g, miRNA-28 and miRNA-502. Currently we evaluate potential association of microRNA expression with the clinical response of MM patients to Bortezomib.In conclusion, our data indicate that miRNAs are differentially expressed in subgroups of MM patients characterized by common cytogenetic abnormalities. Disclosures:Goldschmidt:Johnson and Johnson: Research Funding, Speakers Bureau. Sonneveld:Johnson and Johnson: Research Funding, Speakers Bureau.

HOXB7 Overexpression in Mesenchymal Cells Stimulates the Production of Pro-Angiogenic Molecules: Potential Role in Multiple Myeloma Associated Angiogenesis

Bone marrow (BM) neo-angiogenesis has a critical role in multiple myeloma (MM) progression. It is well established that the angiogenic process in MM is mainly due to an overproduction of pro-angiogenic molecules by MM cells and the BM microenvironment cells. However the molecular mechanisms at the basis of the angiogenic process in MM are currently under investigation. The deregulation of the homeobox genes has been previously associated to tumor progression and neoangiogenesis. Particularly, overexpression of the homeobox HOXB7 is critical in tumor-associated angiogenic switch in solid tumors as breast cancer. Actually the potential role of HOXB7 in MM-induced angiogenesis is not known. In this study we have investigated the expression of HOXB7 by MM and BM microenvironment cells and its potential role in the regulation of the angiogenic process. First, by microarray analysis in a large database of MM patients (n°= 132) we found that HOXB7 was overexpressed by MM cells in about 10% of patients as compared to healthy donors and MGUS subjects. On the other hand HOXB7 mRNA was expressed in 18 out of 23 human myeloma cell lines tested. Moreover, we found that isolated BM mesenchymal (MSC) and osteoblastic (OB) cells, obtained from bone biopsies in a subgroup of MM patients (n°=24) expressed HOXB7 gene by microarray analysis and real time PCR. HOXB7 expression was also investigated at protein level by immunohistochemistry on bone biopsies of MM patients finding that MSC and OB as well as endothelial cells expressed HOXB7 protein mainly at nuclear level. In order to investigate the potential role of HOXB7 in the angiogenic process we enforced HOXB7 expression by lentivirus vectors in MSC using both primary BM MSC and the human MSC cell line HS-5 to obtain a stable transduced cell line. The overexpression of HOXB7 in HOXB7 transduced MSC as compared to the empty vector-transduced MSC cells was confirmed by real time PCR, western blot and immunohistochemistry. By Gene chips U133 plus 2.0 (Affymetrix) we evaluated the gene expression profiling of HOXB7 over-expressing MSC finding that proangiogenic cytokines, metalloproteinases and chemokines were significantly modulated in HOXB7-transduced MSC cells as compared to control cells. Data were validated either by real time PCR or by western blot and by an angiogenesis antibody array showing that bFGF and VEGF production was induced in MSC by HOXB7 overexpression. Consistently, we found that conditioned media of HOXB7-transduced MSC cells significantly stimulated vessel formation as compared to controls using an in vitro angiogenic model. Finally we observed that the angiogenic in vitro differentiation of HOXB7-transduced MSC was significantly increased as compared to controls.In conclusion our data suggest the HOXB7 overexpression in MSC regulates the angiogenic switch and could be a potential therapeutic target in MM-induced angiogenesis.

The AKT/mTOR/P70S6K/4EB-P1 Signaling Pathway Is Activated in a Subset of Multiple Myeloma Patients and Correlates with High Serum Levels of Beta2-Microglobulin

Introduction: The mammalian target of rapamycin (mTOR) is a serine/threonine-specific protein kinase, downstream of the phoshatidylinositol 3-kinase (P13-K/AKT) pathway. Constitutive activation of the mTOR related upstream and downstream effectors including P13-K, AKT, P70S6K and 4E-BP1 have been found in numerous malignancies. Previous studies demonstrated that rapamycin has preclinical potential as therapy for multiple myeloma (MM), especially when associated with other drugs.Methods. We performed immunohistochemical analysis with p-AKT (Ser 473), p-mTOR (Ser2448), p-P70S6K (Thr389) and p-4E-BP1 (Thr37,Trh46) on bone marrow sections of 73 symptomatic MM patients. Mielomatous plasmacells were identified and counted by mouse monoclonal CD138 nd all cases were analyzed using a semiquantitative histologic score (HSCORE) method. Specifically, immunostaining intensity of each case was semiquantitatively scored as follow:0, no staining;1,weak staining;2, moderate staining; and3, strong staining.For each case, a value designed HSCORE was obtained multiplying each intensity with the corresponding percentage of positive cells [HSCORE =∑(1XPC), where 1 and PC represent intensity and percentage of cells, respectively]. Specimen with an HSCORE of ≥30 were classified as p-AKT, p-mTor, p-P706SK and p-4E-BP1 positive. Wilcoxon test was used to compare mTOR expression with clinical data of all patients (including age, presence of bone lesions, isotype, Beta2-microglobulin, haemoglobin, creatinine and albumin serum levels). Common cytogenetic abnormalities (t(11;14), t(4;14), del 13q14 and del p53) were also detected in 61 of 73 (83.5%) patients by FISH analysis on CD138 purified plasma cells.Results. Fouty-four (60.2%) and 46 of 73 (63%) patients stained positive for p –AKT and p-mTOR with a cytoplasmic staining pattern, respectively. P-mTOR immunoreactivity was strongly, moderately and weakly positive in 23.9 %, 34.8 % and 41.3 % of the 46 positive samples, respectively. P-P70S6K and p-4E-BP1 was detected in 53 (72.6%) and 40 (54.8%) patients with a predominantly nuclear staining pattern. The intensity of positivity was distributed as follows: p-P70S6K strongly, moderately and weakly positive, 54.7 (%), 26.4 (%) and 18.9 (%),respectively; p-4E-BP1 strongly, moderately and weakly positive, 62.5 (%), 17.5 (%) and 20 (%), respectively; Of the 46 myelomas stained positive for p-mTOR, 35 expressed p-AKT, 33 expressed p-4E-BP1 and 40 demonstrated p-P70S6K positivity. P-mTOR expression significantly correlated with p-AKT (p=0.003), p-P70S6K (p<0.001) and p-4E-BP1 (p< 0.001) staining consistent with the hypothesis that the AKT/mTOR/P70S6K/4E-BP1 pathway is activated in a subset of MM cases. A significant statistical correlation was found between mTOR expression and serum levels of Beta2-microglobulin ≥3 g/dl (P=0.039). No clearly correlation was found between mTOR pathway activation and the molecular cytogenetic anomalies.Conclusion: We identify a subgroup of MM patients with mTOR pathway activation and poor prognosis. Phosphoprotein staining of the mTOR pathway should be studied further as a means to select patients to receive mTOR inhibitors in combination of chemotherapy. These data also strongly suggest that the AKT/mTOR/P70S6K/4E-BP1 pathway activation may constitute a new biologic parameter regardless of molecular cytogenetic.