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The interaction of multiple myeloma (MM) cells with their microenvironment in the bone marrow (BM) provides a protective environment and resistance to therapeutic agents. We hypothesized that disruption of the interaction of MM cells with their BM milieu would lead to their sensitization to therapeutic agents such as bortezomib, melphalan, doxorubicin, and dexamethasone. We report that the CXCR4 inhibitor AMD3100 induces disruption of the interaction of MM cells with the BM reflected by mobilization of MM cells into the circulation in vivo, with kinetics that differed from that of hematopoietic stem cells. AMD3100 enhanced sensitivity of MM cell to multiple therapeutic agents in vitro by disrupting adhesion of MM cells to bone marrow stromal cells (BMSCs). Moreover, AMD3100 increased mobilization of MM cells to the circulation in vivo, increased the ratio of apoptotic circulating MM cells, and enhanced the tumor reduction induced by bortezomib. Mechanistically, AMD3100 significantly inhibited Akt phosphorylation and enhanced poly(ADP-ribose) polymerase (PARP) cleavage as a result of bortezomib, in the presence of BMSCs in coculture. These experiments provide a proof of concept for the use of agents that disrupt interaction with the microenvironment for enhancement of efficacy of cytotoxic agents in cancer therapy.

Waldenstrom macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow at the time of diagnosis, implying continuous homing of WM cells into the marrow. The mechanisms by which trafficking of the malignant cells into the bone marrow has not been previously elucidated. In this study, we show that WM cells express high levels of chemokine and adhesion receptors, including CXCR4 and VLA-4. We showed that CXCR4 was essential for the migration and trans-endothelial migration of WM cells under static and dynamic shear flow conditions, with significant inhibition of migration using CXCR4 knockdown or the CXCR4 inhibitor AMD3100. Similarly, CXCR4 or VLA-4 inhibition led to significant inhibition of adhesion to fibronectin, stromal cells, and endothelial cells. Decreased adhesion of WM cells to stromal cells by AMD3100 led to increased sensitivity of these cells to cytotoxicity by bortezomib. To further investigate the mechanisms of CXCR4-dependent adhesion, we showed that CXCR4 and VLA-4 directly interact in response to SDF-1, we further investigated downstream signaling pathways regulating migration and adhesion in WM. Together, these studies demonstrate that the CXCR4/SDF-1 axis interacts with VLA-4 in regulating migration and adhesion of WM cells in the bone marrow microenvironment.

Waldenstrom macroglobulinemia (WM) is an incurable low-grade lymphoma characterized by bone marrow (BM) involvement of IgM secreting lymphoplasmacytic cells. The induction of unfolded protein response (UPR) genes (“physiologic” UPR) enables cells to differentiate into professional secretory cells capable of production of high amounts of endoplasmic reticulum (ER)–processed proteins, such as immunoglobulins. Ultimately, the initially cytoprotective UPR triggers an apoptotic cascade if ER stress is not corrected, called proapoptotic/terminal UPR. We show that WM cells inherently express the physiologic UPR machinery compared with normal BM cells, and that increased ER stress leads to proapoptotic/terminal UPR in WM cells. We therefore examined tunicamycin, ER stress inducer, for potential antitumor effects in WM. Tunicamycin induced significant cytotoxicity, apoptosis and cell-cycle arrest, and inhibited DNA synthesis in WM cell lines and primary BM CD19+ cells from patients with WM with an inhibitory concentration (IC50) of 0.5 μg/mL to 1 μg/mL, but not in healthy donor cells. Importantly, coculture of WM cells in the context of the BM microenvironment did not inhibit tunicamycin-induced cytotoxicity. Finally, we demonstrate that ER stress inducer synergizes with other agents used in the treatment of WM. These preclinical studies provide a framework for further evaluation of ER stress inducing agents as therapeutic agents in WM.

Waldenström macroglobulinemia (WM) is an incurable low-grade B-cell lymphoma characterized by high protein turnover. We dissected the biologic role of the proteasome in WM using 2 proteasome inhibitors, NPI-0052 and bortezomib. We found that NPI-0052 inhibited proliferation and induced apoptosis in WM cells, and that the combination of NPI-0052 and bortezomib induced synergistic cytotoxicity in WM cells, leading to inhibition of nuclear translocation of p65NF-κB and synergistic induction of caspases-3, -8, and -9 and PARP cleavage. These 2 agents inhibited the canonical and noncanonical NF-κB pathways and acted synergistically through their differential effect on Akt activity and on chymotrypsin-like, caspaselike, and trypsinlike activities of the proteasome. We demonstrated that NPI-0052–induced cytotoxicity was completely abrogated in an Akt knockdown cell line, indicating that its major activity is mediated through the Akt pathway. Moreover, we demonstrated that NPI-0052 and bortezomib inhibited migration and adhesion in vitro and homing of WM cells in vivo, and overcame resistance induced by mesenchymal cells or by the addition of interleukin-6 in a coculture in vitro system. Theses studies enhance our understanding of the biologic role of the proteasome pathway in WM, and provide the preclinical basis for clinical trials of combinations of proteasome inhibitors in WM.

Aim Allogeneic hematopoietic stem cell transplantation (HSCT) is increasingly used for the treatment of various hematological conditions with poor outcome. However, despite its effectiveness opportunistic infection, graft versus host disease (GVHD), drug toxicity and relapse frequently lead to life threatening complications and 15-20% of HSCT recipients ultimately require life sustaining therapies. Nowadays, a trend toward the use of less toxic conditioning regiments, different graft sources and better management of GVHD is observed. Also recent finding emphasize advances in the ICU management and triage policies of hematological patients. However, it is not known whether these changes altered the typology and outcomes of HSCT recipient admitted to ICU. We then performed a multicenter study to assess the impact of these changes. Methods All adults admitted in 3 ICU in Paris France from 1997 to 2011 were included if they previously received an HSCT. Data from medical charts were retrospectively retrieved and analyzed after the patients gave written informed consent. 497 patients were included, among whom 209 were admitted in the 1997-2003 period (this cohort was already published)1 and 288 in the 2004-2011 period. This corresponded to 2286 HSCT procedures over the same centers and period of time. Patients, HSCT characteristics and outcomes of the 2 cohorts were compared and prognostic factors analysis was performed in the recent period cohort. Results Over the time, HSCT recipients were more likely to be older (48 vs. 41 years old p 2, mechanical ventilation and renal replacement therapy were independently associated with day 90 mortality. Conclusion Despite improvement of ICU care in HSCT patients with no GVHD, this study show that life-sustaining therapy still remains mostly unsuccessful when used in patients with GVHD and deep immunosuppression. These data argue for a more rational policy of ICU admission triage in HSCT recipient 1Pene, Aubron, Azoulay et al. J. Clin. Oncol. February 1, 2006; 24(4): 643 - 649. Disclosures: No relevant conflicts of interest to declare.

Background. Measurement of serum M-spike is used to determine response to therapy and treatment free survival in Waldenstrom Macroglobulinemia (WM). However, there are many limitations to the use of IgM M-spike, and new sensitive markers are needed to determine early response or progression in these patients. We have previously shown that involved serum free light chain (sFLC) accurately diagnosed patients with WM, and correlated with markers of poor prognosis, specifically beta- 2 microglobulin (B2M). In this study, we sought to determine the role of levels of involved in the response to therapy in patients with WM treated on clinical trials, and compare it to the response observed using the traditional M-spike measurement. Methods. We prospectively studied involved sFLC in 61 WM patients enrolled on 2 clinical trials, at diagnosis (N=8) and with relapse/refractory disease (N=53). Patients were treated on one of two clinical trials: either perifosine (N=30; given 150mg oral daily) or combination of bortezomib and rituximab (N=30; given IV bortezomib 1.6mg/m2 at days 1, 8, 15 q 28 days × 6 cycles and rituxan 375 mg/m2 at days 1, 8, 15, 22 on cycles 1 and 4). Response was assessed after cycle 2, confirmed on 2 consecutive measurements, and included minor response (MR), partial response (PR) or complete remission (CR). Results. The baseline characteristics of the patients were as follows: the median age was 65 years (44–83), male/female ratio 2.38, serum B2M 3.0mg/L (1.00–7.30), hemoglobin 11.0g/dL (7.00–14.90), platelet count 216 ×109/mm3 (46–563), serum M-spike 2.24g/L (0.41–4.62), and involved sFLC 95.2mg/L (4.92–868). The WM International staging system (WM-ISS) showed that 28% of patients had low ISS, 31% intermediate, and 41% high ISS scores. The overall response rate for the entire cohort was 62.3%, assessed by M-spike measurement (MR=23, PR=13, nCR=2). The overall response rate using involved sFLC level was 72.1% (MR=14, PR=29, and CR=1), (p Conclusion. In this study, we identified that involved sFLC is a new and reliable marker for monitoring response to therapy in WM disease, especially to determine early response in these patients.

Background. Multi-level genetic characterization of Waldenstrom’s Macroglobulinemia (WM) is required to improve our understanding of the underlying molecular changes that lead to the initiation and progression of this rare disease. Cytogenetic and molecular studies using gene expression analysis at the mRNA level have demonstrated minimal changes in WM cells. The most commonly identified abnormality in WM is the deletion of the 6q arm. Karyotype studies showed this abnormality in 16% of WM patients, but recent studies have shown that this deletion is present in >50% of patients using cIgMFISH. We therefore sought to investigate molecular changes that occur at the level of the “transcriptome”, indicating genes that have been actively transcribed by PolIymerase II (Poll II) enzyme using ChIP on chip technology. This technique offers the advantage of discovery of genes deregulated in WM with greater confidence than when cDNA (from RNA) is hybridized to tiling arrays. Methods. Experiments were performed using genomewide location analysis with ChIP-on-Chip TranscriptionPath technology by Genpathway (San Diego, CA). TranscriptionPath is a novel assay that identifies and quantifies genomic DNA sequences undergoing active transcription within cells. We studied CD19+ selected WM samples (N=7) and CD19+ control cells obtained from normal bone marrows (N=2). The chromatin was immunoprecipitated using an antibody specific for pol II. DNA was labeled using the Affymetrix GeneChip® WT Double-Stranded DNA Terminal Labeling Kit and hybridized to the human promoter tiling arrays. Raw data from the scans were analyzed using Tiling array analysis algorithm MAT (Johnson et al., 2006) to determine the difference in Pol II binding sites/activities using a bandwidth of 300bp and p value cut off of . RefSeq mapping were performed using Galaxy (Giardine et al., 2005). Gene ontology analysis was conducted using DAVID at http://david.adcc.ncifcrf.gov/. Results: Analysis showed that most samples with WM demonstrated a higher level of transcribed genes compared to normal control. Overall, there were 9304 regions corresponding to 4975 known genes (7405 unique Genebank accession) in the WM samples that had enriched Pol II binding activities compared to normal control. We then focused on the long arm of chromosome 6 to determine changes that occur at the “transcriptome”. We did not analyze the samples for 6q deletion to avoid bias in the analysis. At p-value of level, 193 regions were identified with higher Pol II binding activities in WM patients compared with normal donor. With at least 100 bp overlap, the regions hit 144 genes (144 unique Gene Bank accession), which contained 106 unique genes. Of these, proteasome subunit beta type 1, ubiqutin-conjugating enzyme E2, mitogen activated protein kinase, cyclin C, transcription factor B1, BCL2-associated transcription factor 1, ribosomal protein 12, wilms tumor 1 associated protein (WT1), Forkhead Box O3A (FOXO), CBP/P300-interacting transactivator, Interferon gamma receptor and chemokine receptor CCR6 were among the most overtranscripted genes observed in WM compared to control. In addition, poly(a)- specific ribonuclease (parn)-like domain containing 1 (PNLDC1) showed decreased Pol II binding activity in WM compared to control. Conclusion: TranscriptionPath ChIP on Chip/ Tiling provides the first assay for comprehensive identification of sequences undergoing active transcription across the genome in WM with high sensitivity and specificity. These included both known genes such as regulators of the proteosome/ubiqutin pathways, MAPK pathways, and Bcl-2, and previously unknown genes such as FOXO and WT1. Translocation of FOXO with the MLL gene is associated with secondary acute leukemia. Similarly, WT1 has been associated with leukemias. Interestingly, we did not identify a significant number of genes underexpressed in WM samples compared to control in the 6q. Further confirmation of the role of these genes in the regulation of tumor progression in WM is underway.

BACKGROUND: Multiple myeloma (MM) is a plasma cell malignancy that depends on interactions with the bone marrow (BM) microenvironment for growth and survival. In turn, adhesion of MM cells to the BM stroma provides a mechanism of resistance from standard chemotherapeutic agents. Recently, our lab has shown that by disrupting this adhesion using a selective CXCR4 inhibitor named AMD3100, MM cells are more sensitive to the proteasome inhibitor Bortezomib (Ghobrial lab, unpublished data). CXCR4 has been a particularly attractive target because its ligand SDF-1 is known to induce p42/44 MAPK, AKT, and the down-stream anti-apoptotic protein bad in MM cells, leading to increased MM growth and survival. Until recently, CXCR4 was thought to be a canonical receptor for the SDF-1 ligand. However, a second chemokine receptor for SDF-1 was subsequently discovered and named CXCR7. CXCR7 is a novel chemokine receptor that is important in cell adhesion, growth and survival in several tumor types. However, the role of CXCR7 in multiple myeloma (MM) has yet to be explored. Furthermore, the ability of SDF-1 ligand to regulate MM function via CXCR7 has not been studied. METHODS: The MM cell lines (U266, MM1.S, RPMI, OPM2, OPM1) were used. After informed consent was obtained, primary bone marrow samples from MM patients were collected. CD138 positive mononuclear cells were isolated by microbead selection. The expression of CXCR7 on MM cell lines and patient samples was confirmed using flow cytometry and RT-PCR analysis. For functional in vitro and ex-vivo assays, the CXCR7 selective antagonist 733 was used (ChemoCentryx Inc., Mountain View, CA). RESULTS: Here we show that CXCR7 was expressed on all tested MM cell lines and primary patient samples as demonstrated by flow cytometry and RT-PCR. Furthermore, CXCR7 was found to regulate SDF-1 induced MM cell adhesion, as demonstrated by in vitro assays using a small molecule compound specific for CXCR7 (733). The CXCR7 antagonist showed significant inhibition of adhesion of MM cell lines and patient samples to fibronectin, endothelial cells and stromal cells, with 50% reduction of adhesion at 5nM of the CXCR7 inhibitor, and with similar activity compared to 20uM of AMD3100 (CXCR4 inhibitor). However, unlike CXCR4, CXCR7 did not effect trans-well migration to SDF-1 chemokine. Interestingly, both receptors were found to be important for trans-endothelial migration of MM cells. Moreover, pre-treatment with 733 reduced homing of MM cells to the BM niche in vivo. Previous studies have failed to show signaling in response to CXCR7 in many tumor types. Here, we demonstrate that treatment with 733 inhibited SDF-1 induced pERK and pAKT, ribosomal pS6Kinase, pGSK3, pSTAT3, pFAK and pPAK signaling pathways, confirming a role for CXCR7 in facilitating SDF-1 signaling. This effect was further confirmed using immunofluorescence. To investigate whether CXCR7 and CXCR4 interact directly, we examined the effect of 733 and AMD3100 on CXCR4 expression and found that AMD3100 significantly inhibited CXCR4 expression, while 733 had no effect on CXCR4 expression, even in the presence of SDF-1. The CXCR7 inhibitor had no effect on the survival of MM cells using MTT and flow cytometry analysis, while high doses of 733 (1uM) had modest inhibition of proliferation. Interestingly, 733 prevented the growth advantage induced by 30nM SDF-1 at 24 hrs. CONCLUSION: Together, these results demonstrate the importance of CXCR7 in regulating MM adhesion and homing, and highlight the differential effects of CXCR4 and CXCR7 in regulating SDF-1 signaling in MM, thus providing a rationale for targeting the SDF-1/CXCR7 axis in MM.

Background: WM is an incurable low-grade lymphoplasmacytic lymphoma. Bortezomib has recently demonstrated about 50% ORR in patients with relapsed WM. We therefore investigated the in vitro effect of the new proteasome inhibitor NPI-0052 (N) alone and in combination with Bortezomib (B). Methods: WM cell lines (BCWM1,WSU-WM) and IgM secreting cell lines (MEK1, Namalwa) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured using MTT assay and [3H]-thymidine uptake. Determination of the synergistic effect [combination index (CI)] of combination was calculated using the CalcuSyn software. Cell signaling and apoptotic pathways were determined by Western Blot. We also tested the effect of N on WM cells in the co-culture with BMSCs. Activity of the 20S proteasome was determined by detecting the release of the fluorophore AMC, after cleavage from the labeled substrates specific for each enzymatic activity. Results: N induced cytotoxicity and inhibition of DNA synthesis (IC50 15nM) in BCWM.1 (48 h). Similar effects were demonstrated in IgM secreting cell lines and primary CD19+ WM cells (IC50 18–30nM). No cytotoxicity was observed on peripheral blood mononuclear cells. The combination of N+B significantly inhibited BCWM.1 proliferation compared to each agent alone: B (5nM) induced cytotoxicity in 8.5%, which increased to 26%, 40% and 53% in the presence of N 2.5nM (CI:0.83), 5nM (CI:0.72) and 10nM (CI:0.7) respectively, indicating synergism. To determine the mechanism of synergy, we investigated the effect of the two agents and their combination on proteasome activity and on signaling pathways, specficially the Akt pathway. Both N and B inhibited the three proteasome activities: the combination of N+B was increased compared to the effect of each agent alone on the caspase-like (C-l) activity of the proteasome. B and N used as single agents induced 29% and 34% inhibition of the C-l activity, respectively, compared to 60% when B and N were used in combination. The C-l activity preferentially cleaves substrates with an aspartic residue in P1 position, like the substrates of the caspases. N induced caspase-8, PARP cleavage and increase of Smac as well as down-modulation of the anti-apoptotic proteins c-IAP, XIAP, survivin, Bcl-2, Mcl-1. The combination of N+B induced a stronger and more significant induction of caspase-8, -PARP cleavage, as well as caspase-3 and -9, which were not affected by using N alone. Similarly, Smac modulation resulted in a more significant induction when cells were exposed to both proteasome inhibitors. N inhibited Akt phosphorylation in BCWM.1 cells (6h) in a dose-dependent manner. GSK3 phosphorylation and ribosomal protein-S6, Akt-downstream target proteins, were also markedly inhibited. Importantly, N inhibited Akt phosphorylation and Akt activity in BCWM.1, even when combined with B, which induced increase of Akt phosphorylation. Lastly, neither exposure to IL-6 nor adherence to BMSCs conferred protection to WM cells against NPI-induced cytotoxicity. Conclusion: NPI-0052 has significant antitumor activity in WM in vitro especially in combination with Bortezomib. These results provide the framework for clinical trials in WM.

Background: Multiple Myeloma (MM) and Waldenstrom Macroglobulinemia (WM) are characterized by widespread involvement of the bone marrow (BM) as the result of successful homing, engraftment and growth of tumor cells at that site. The receptor for SDF-1, CXCR4 has already been implicated by us and others in the migration and homing of tumor cells to the bone marrow. Recently, a new receptor for the chemokine SDF-1 has been described, namely CXCR7. To date, it has been implicated in the migration of embryonic cells during neurogenesis in zebrafish. It has been reported to be expressed in cancer cells but its function remains unknown. We have previously shown that disruption of the CXCR4/SDF-1 axis interferes with homing of MM cells to the BM. To better understand the homing and migration of MM and WM tumor cells, we sought to study the expression and function of CXCR7. Methods: MM (U266, MM1.S, RPMI, OPM2, OPM1, KMS12BM) and WM (BCWM.1 and WSU-WM) cell lines were used. CD19+ and CD138+ cells were extracted from patient bone marrow samples by microbeads selection after informed consent. To determine the expression of CXCR7 on MM and WM cell lines as well as primary samples, we used flow cytometry and RT-PCR analysis. The migration of tumor cells towards SDF-1 was studied using the transwell boyden chamber migration assay. The adhesion of tumor cells to fibronectin using a fluorometric assay. The CXCR7 inhibitor CCX-754 (ChemoCentryx Inc., Mountain View, CA) was used. Results: CXCR7 was expressed in all cell lines tested except WSU-WM by flow cytometry and RT-PCR. Interestingly, U266 cell line did not express surface CXCR4 and expressed CXCR7 and therefore was used in further experiments to determine the differential role of CXCR7 in SDF-1 related function. Primary WM (n=14) and MM (n=7) cells expressed low intensity CXCR7 by flow cytometry. Use of the above cells in experiments utilizing small molecule antagonists of both CXCR4 and CXCR7 suggested that inhibiting the binding of SDF-1 to one receptor could impact the signaling functions of the other. Experiments are ongoing to clarify the nature of the interaction between these two SDF receptors. Furthermore, adhesion of U266 cells to fibronectin was increased in presence of SDF-1, and inhibited in the presence of CCX-7754. Further analysis to examine CXCR7 and CXCR4 antagonism in adhesion are ongoing. Conclusion: we showed for the first time CXCR7 expression on MM and WM tumor cells. Our results lead us to conclude that through binding of a shared ligand, CXCR7 and CXCR4 may modulate the biological activity of the other.

Background. Waldenstrom Macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow (BM). Adhesion of malignant cells to the BM microenvironment induces proliferation and resistance to therapy. We previously showed that SDF-1/CXCR4 axis regulates migration and adhesion of WM cells in vitro indicating a potential role in homing. Homing of malignant cells to the BM niches requires active navigation through the endothelial cell barrier where tumor cells must adhere, roll and transmigrate. We hypothesized that CXCR4 and its interaction with VLA-4 is critical for this 3-step process. Methods. The level of CXCR4 and VLA-4 was determined using flow cytometry and RT-PCR in patient samples and WM cell lines (BCWM.1 and WM-WSU). The CXCR4 inhibitor AMD3100 (10–100uM, Sigma, MO), Gi protein inhibitor pertussis toxin, PTX (10–200ng/ml, Sigma, MO) were used to inhibit CXCR4 signaling. These studies were confirmed using CXCR4 knockdown using shRNA with lentivirus infection. Adhesion assay was done with either plates coated with the VLA-4 ligand fibronectin for adhesion of tumor cells in presence and absence of SDF1 (30nM), or coated with endothelial cells (HUVEC) and BMSCs for cell-cell adhesion experiments. Transmigration assay was performed using the transwell system with either SDF1 in the lower chamber to study migration towards SDF1, or with the upper chamber coated overnight with endothelial cells (HUVEC) and BMSCs to study transmigration. Experiments with HUVEC cells were performed in absence or presence of TNF-alpha that activates the HUVEC cells. Results. WM tumor cells from patients and cell line expressed high surface levels of CXCR4 (mean 70%) and VLA-4 (mean 95%). Adhesion of WM cells to fibronectin was significantly increased with SDF1 compared to BSA control, and AMD3100 20uM inhibited adhesion by 50% compared to control. Similar results were obtained with PTX 200ng/mL and using CXCR4 knockdown in WM cells. Furthermore, WM cells adhesion was greatly enhanced in co-culture with BMSCs and endothelial cells, and was significantly inhibited using AMD3100 and PTX. To further dissect the mechanisms of interaction of CXCR4 and VLA-4, we showed that AMD3100 inhibited surface expression of CXCR4 but not of VLA-4. Immunoprecipitation studies showed that CXCR4 and VLA-4 directly interact in response to SDF-1. Downstream signaling pathways showed that CXCR4 regulates migration and adhesion through the PI3K/Akt and ERK/MAPK pathways. We next studied whether CXCR4 was involved in transmigration through the endothelial barrier. We demonstrated that BCWM.1 cells transmigrated through the barrier of endothelial cells, that was increased when SDF1 was added to the lower chamber, and that AMD3100 inhibited the transmigration of BCWM.1 cells. Studies of the role of CXCR4 in rolling on endothelial cells is ongoing. Conclusion. We showed CXCR4/SDF1 axis regulates migration and adhesion of WM cells to the BM microenvironment indicating a potential role in homing. Moreover, we demonstrate that CXCR4/SDF1 axis is critical for transendothelial migration, a critical step of homing and egression of tumor cells in and out the BM niches.

We have previously demonstrated that a consistent feature of malignant plasma cells of multiple myeloma (MM) is the aberrant expression of genes important in patterning and development, such as members of Hedghehog (Hh) pathway (FE Davies et al, Blood 2003). These findings suggest that overexpression of genes of this pathway, already involved in many solid tumors and recently implicated in maintaining a proposed MM stem cell compartment (CD Peacock et al, PNAS 2007), might be one of the mechanism through which Hh-signaling contributes to tumorigenesis in MM. Therefore, several small molecule modulators of Hh-pathway, which work as agonists and antagonists, are currently under development. We evaluated, by microarray analysis, the expression of Hh pathway genes in MM cell lines and primary MM cells vs. plasma cells from healthy donors. We found that primary MM cells overexpress Sonic (Shh), Smoothened (Smo), Patched (Ptc), Gli-1 and Gli-3 (relative expression ratios ranging from +1.8 to +5.0). Overexpression of Patched was also observed in most of the MM cell lines analyzed (+5.0 ratio in 5 of 6 MM cell lines). Additionally, we confirmed the expression of Shh and of Gli-1, by flow cytometry and western blotting respectively, in a large panel of MM cell lines. These data suggest an activation of the Hh-pathway in MM that, in some cell lines, is Shh-dependent. Therefore, we investigated the therapeutic potential of Hh-inhibitors in MM. We assayed the cell viability and proliferation, by MTT and Thymidine uptake respectively, in 8 MM cell lines after 72 hours of treatment with the small molecule Smo-inhibitor CUR-0199691 (Genentech). We observed a reduction in MM cell viability, with IC50 values ranging between 4.5–9.5 μM in these 8 cell lines and an inhibition of MM cell proliferation with IC50 values ranging between 0.5 and 2.5μM in the same cell lines. MM cell sensitivity to this compound appears to be related to the level of expression of Gli-1, since the cell lines with lower level of expression of Gli-1 were more sensitive. The treatment of these MM cell lines with Cyclopamine, another Hh-inhibitor, showed an IC50 between 7.5μM and 10μM after at least 96 hours of treatment in 4 of the MM cell lines tested. CUR-0199691 is also active in primary MM cells, triggering inhibition of proliferation by 50% at 5μM after only 24h of treatment, while cyclopamine reduces MM cell proliferation (normalized to the effect of tomatidine, its inactive analog) by 30% at 20μM after a 48 hour treatment. Annexin V-PI staining of Hh inhibitor-treated KMS11 cells, which are one of the most sensitive MM cell lines, showed induction of apoptosis, evidenced by detection of 12 and 15% of MM cells being Annexin V+/PI- after 48h and 72h respectively with 5μM of CUR-0199691. These results, taken together, show that the Hh-pathway is fuctionally active in MM and that the novel Hh pathway inhibitor CUR-0199691 is 4–5 times more effective than cyclopamine in both MM cell lines and primary MM cells. These studies provide the framework for further preclinical evaluation of CUR-0199691 in MM models towards possible future clinical trials.

Background: Resveratrol is a polyphenolic natural product, synthesized by a wide variety of plant species including grapes. It has gained considerable attention because of its anti-cancer properties, as demonstrated in solid and haematological malignancies. We therefore examined Resveratrol for its anti-tumor activity in Waldenstrom’s Macroglobulinemia (WM). Methods: We examined the effect of increasing concentrations of resveratrol (5–80 μM) on WM cell lines (BCWM.1), IgM secreting low-grade lymphoma cell lines (WM-WSU, MEC-1, RL), peripheral blood mononuclear cells (PBMCs) isolated from healthy donors, primary CD19+ WM cells and bone marrow stromal cells (BMSCs) isolated from bone marrow of patients with WM. [3H]-thymidine uptake and calcein-AM assay were used to evaluate the effect of resveratrol on proliferation and cytotoxicity respectively. Apoptosis and cell cycle analysis were investigated at 24h by flow cytometry using Annexin V-propidium iodide (PI) staining and PI-staining respectively. Apoptotic and cell signaling pathways targeted by resveratrol were investigated by Western Blot at 24 h and 6 h respectively. Since BMCSc confer growth and resistance to conventional treatments, we also tested the effect of resveratrol on WM cells co-cultured with BMSCs. Gene expression analysis has been performed on BCWM.1 cultured in presence or absence of resveratrol. Results: Resveratrol induced significant cytotoxicity and inhibition of DNA synthesis at 24 and 48 h on BCWM.1 with an IC50 of 10–20μM. Similar data was obtained with primary CD19+ WM cells. In contrast, resveratrol did not trigger significant reduction of proliferation of PBMCs. Resveratrol induced apoptosis in BCWM.1, as demonstrated by flow cytometry. Dose-dependent apoptosis at 24h with induction of JNK followed by caspases 3, 8, 9 and PARP cleavage was also observed. Resveratrol induced reduction of Mcl-1 and increase of p53, p63 and p73, as shown by gene expression analysis and western blot, providing an alternative mechanism of cell growth arrest in absence or mutation of p53. In parallel, resveratrol induced down-regulation of cyclin-D1, -D2, -E1, cdk-2, -4, -6 and up-regulation of p21Cip1 and p27Kip1, demonstrated in terms of transcript by gene expression analysis and protein levels by western blotting. We next observed that resveratrol inhibited ERK and Akt phosphorylation in BCWM.1 in a dose-dependent manner, as well as Akt activity, as shown by the in vitro Akt kinase assay. Phosphorylation of GSK3α/β and ribosomal protein-S6, downstream target proteins of Akt, were also markedly inhibited. Resveratrol also down-regulated Wnt signaling pathway with a reduction of nuclear β-catenin levels and a decrease of myc and survivin, both downstream target proteins of β-catenin. Lastly, adherence to BMSCs did not confer protection to WM cells against resveratrol-induced cytotoxicity Furthermore, resveratrol demonstrated synergistic cytotoxicity when combined with dexamethasone, fludarabine and bortezomib. Conclusion: These in vitro data demonstrated that resveratrol has significant antitumor activity in WM, providing the framework for clinical trials in WM patients.

Background: The NF-kB pathway has been implicated in tumor survival and growth, and to induce resistance to conventional agents. Because of the natural ability of tumor cells to overcome single agent antitumor activity, we hypothesized that targeting differentially NF-kB pathway with combination of proteasome inhibitor bortezomib and of Akt inhibitor perifosine might lead to synergistic cytotoxicity on WM. Methods: The WM cell line BCWM.1 was used in these studies. Primary CD19+ WM cells were obtained from the bone marrow (BM) of patient after informed consent. Perifosine (P) was provided by Keryx (NY), Bortezomib (B) from Millennium (MA) and Rituximab (R) from Genentech (CA). NF-kB was studied using the Chromatin Immunoprecipitation (ChIP)-based assays, a new technology that allows for the specific analysis of activation/inhibition of one specific gene in the entire genome. IkB gene was used in this study as NF-kB target-activation gene. Quantitative real-time PCR (qPCR) for IkB in the p65NF-kB-DNA immunoprecipitated fragments was assessed and those results were confirmed at the transcriptional level using qPCR and immunoblotting on cytoplasmic/nuclear fractionation of cells. NF-kB activity assay was confirmed using the DNA-binding ELISA-based assay Active Motif kit on nuclear extract. Antibody-dependent cellular cytotoxicity assays (ADCC) was performed in presence of rituximab or human control IgG1. Results: We first showed that perifosine and bortezomib combination [P+B] induced synergistic cytotoxicity and inhibition of proliferation in WM cells. Similar cytotoxicity was observed in primary patient tumor cells and in presence of BM microenvironment, but spared normal hematopoietic cells. We next demonstrated that P and B inhibited p65NF-kB nuclear translocation and downstream IkB target gene using ChIP, and that the combination [P+B] showed additive inhibitory activity. We next sought to further dissect molecular mechanisms of synergy induced by the combination. B 5-10nM inhibited phosphorylation of ERK MAPK pathway and slightly upregulated Akt activity. On the other hand, P 10uM inhibited Akt phosphorylation, but induced activation of the ERK MAPK pathway. Interestingly, while either agent differentially upregulated one of those 2 signaling pathways, their combination [P+B] was able to overcome resistance induced by the other agent. This was demonstrated by significant inhibition of downstream target proteins of Akt, and by a significant decreased phosphorylation of fusion protein GSK3a/b using an in vitro Akt kinase assay. We next sought to examine the combination of R with [B+P], since R is known to inhibit Akt and NF-kB pathways. Pretreatment of WM cells with P, B or the combination [P+B] led to a significant increase in the ADCC cytotoxicity of R compared to R alone (p=0.025). We next confirmed that the combination [P+B+R] inhibited NF-KBp65 function in WM cells, using Active Motif assay and ChIP for IkB. Conclusion: Together these studies provide the framework for clinical studies of combination of perifosine with bortezomib and rituximab in a sequential approach to improve patient outcome in WM.

Background: MM is characterized by widespread involvement of the bone marrow (BM) as the result of successful homing, engraftment and growth of myeloma cells. The BM provides protection and resistance of MM to therapeutic agents. Therefore, disruption of the interaction of MM cells with their microenvironment should lead to enhanced sensitivity to therapeutic agents. We hypothesized that disrupting CXCR4/SDF-1 axis will induce mobilization of MM cells from the BM into the circulation. Methods: MM.1S cells were co-cultured with bone marrow stromal cells (BMSCs) in the presence of AMD3100 (50uM, Sigma), bortezomib (0–2.5nM, Millennium) or combination of both; and cell proliferation was measured using [3H]-thymidine uptake. We then tested the in vivo AMD3100-induced mobilization of MM cells after they homed to the BM. MM1.S cells that had been fluorescently labeled with DiD (Invitrogen) were injected into mice through their tail veins. Beginning 24 hours later and for three subsequent days, the mice were treated with 5mg/kg AMD3100 sq daily, injected with fluorescently labeled c-kit antibody for HSC detection and immediately monitored for presence of circulating MM1-S cells or HSCs, using in vivo flow cytometry. To test whether AMD3100 induces mobilization of MM cells in established tumors, a GFP+ and luciferase+ osteotropic MM.1S cell line (Luc+GFP+MM.1S) was developed. This mouse model was used for continuous, real-time quantitation of MM cells mobilization. Bioluminescence imaging was used to determine tumor growth in vivo. Mice were treated with AMD3100 (5mg/kg, daily), bortezomib (1mg/kg biweekly) or the combination. Results: There was a significant increase in proliferation of MM cells in co-culture with BMSCs compared to MM cells alone. Moreover, AMD3100 alone did not inhibit proliferation; however, it significantly enhanced the cytotoxic effect of bortezomib in the presence of stromal cells. In vivo studies revealed that, unlike HSC, no appreciable mobilization of MM1S cells occurred after the first AMD3100 injection; however, the second AMD3100 treatment induced a fourfold increase in circulating MM cell numbers above background, while no further remarkable increase in circulating HSC was observed. The effect of AMD on an established tumor model showed that by the end of the first week of AMD3100 or bortezomib treatment, a 2-fold increase of circulating MM cells was observed compared to control mice, a trend which continued for three weeks. Moreover, compared to AMD3100 or bortezomib treatment alone, a further increase of circulating MM cells was observed in the peripheral blood of mice treated with combination of AMD3100 and bortezomib following the second bortezomib treatment. In the AMD3100 only treated mice, the counts of circulating MM cell continued to increase and tumors continued to progress, while in the AMD3100-bortezomib-treated mice, the circulating MM cell count decreased and the tumors regressed. Conclusion: These data support the hypothesis that disruption of the CXCR4/SDF-1 axis mobilizes MM cells, and that AMD3100 can be used to enhance the effects of therapeutic agents such as bortezomib.

Background. Several TNF family members (CD40L and BAFF/BLYS) have been implicated in Waldenstrom’s Macroglobulinemia (WM) cell growth and survival. More recently, abnormalities in the APRIL-TACI pathway have been demonstrated by us in WM cells (Hunter, ASH2006, #228). TRAFs (TNFR-associated factor) are a family of adaptor proteins that mediate signal transduction from multiple members of the TNF receptor superfamily. In particular, TRAFs facilitate pro-apoptotic signaling from the TACI receptor, and TRAF2 is of importance among the TRAF adapter proteins since this protein is required for TNF-alpha-mediated activation of SAPK/JNK MAPK known to be involved in drug-induced death of tumor B cells. We therefore examined the role of TRAF2 in WM growth and survival. Method. We investigated TRAF2, 3 and 5 gene expression in WM patient bone marrow (BM) CD19+ cells and cell lines (BCWM.1, WSU-WM) and compared their expression to BM CD19+ cells from healthy donors. Expression of human TRAF transcripts were determined using real time quantitative RT-PCR (qPCR) based on TaqMan fluorescence methodology. To evaluate the role of TRAF2, a knockdown model was prepared in BL2126 B-cells and BCWM.1 WM cells using electroporation, with resulted ≥50% knockdown efficiency using RT-PCR and immunoblotting. Results. We found that TRAF3 and 5 gene expression was higher in WM versus healthy donors, while TRAF2 expression was lower in 8/13 (60%) patients, using qPCR. TRAFs gene expression did not correlate with tumor burden or WM prognostic markers. We next sought to understand the biological sequelae of TRAF2 deficiency in BL2126 and BCWM.1 cells and found that TRAF2 knockdown induced increased survival at 72 hours in both cell lines. We next studied sequence analysis of 20 WM patients CD19+ BM cells to determine whether there was a TRAF2 genomic alteration, and found heterozygous early termination mutation in exon 5 in 1 (5%) patient. Conclusion. Our data demonstrate that TRAF2 is a commonly dysregulated TNF family adapter protein in patients with WM, with important consequences in WM cell growth and survival.

Background. WM is characterized by excess secretion of IgM in the serum. Response to therapy is assessed according to the Consensus panel recommendations with the use of IgM by serum protein electrophoresis (M spike). However, there are many limitations to the use of M-spike level, and new markers are needed. We, and others, have recently demonstrated that the measurement of involved sFLC values accurately identified patients with poor prognostic features of WM. We sought to determine whether involved sFLC level can be used as a reliable and sensitive marker to study response to therapy in WM. Methods. We prospectively studied M-spike and involved sFLC levels in 32 patients with relapse/refractory WM, treated on two phase II clinical trials, single agent perifosine (N=21; given 150mg oral daily for 28 days), or bortezomib-rituximab (N=13; given IV bortezomib 1.6mg/m2 at days 1, 8, 15 q28 days x 6 cycles and rituximab 375mg/m2 at days 1, 8, 15, 22 on cycles 1 and 4). Levels of sFLC and M spike were measured on day 1 of each cycle. In addition, sFLCs were measured weekly for the first month in the bortezomib-rituximab study (N=12). Responses were assessed after 2 cycles of therapy. Time to response was determined from initiation of therapy to the time of initial response. Results. Characteristics in the overall population were not different between the 2 groups treated, and median values were: beta-2 microglobulin (B2M) 2.7mg/L, hemoglobin level 11g/dL, serum IgM 40g/L, M-spike 2.4g/dL, and involved sFLC 111mg/L. The median follow-up was 5 months, and was not significantly different between the 2 trials. The overall response rate (ORR) of the bortezomib/rituximab clinical trial was 85% and the perifosine clinical trial was 33%. The ORR as calculated by the sFLC in the bortezomib-rituximab trial was 100% and in the perifosine trial was 39%. Eleven (34%) patients achieved better responses by sFLC compared to M spike. Of these, 7 were classified as minor response (MR) by M spike but achieved a partial response by sFLC, and 4 were classified as stable disease by M-spike, but achieved MR using involved sFLC. Only 2 patients (6%) had a better response using M spike compared to sFLC. The overall median time to response was 79 and 110 days using M-spike, and 68 and 41 days using sFLC, in bortezomib-rituximab and perifosine trials, respectively. We next found that neither M-spike nor involved sFLC measured prior to therapy could predict response. We then investigated whether sFLC could predict response to therapy using weekly sFLC for the first month. Among the 14 patients studied, 7 showed a decrease in sFLC within the first month, which predicted response by using M spike. On the other hand, the other 7 patients had an sFLC “flare” within the first 4 weeks of therapy, and these patients had a delayed response to therapy using M spike criteria. Conclusion. Involved sFLC is a sensitive marker for monitoring response to therapy in patients with WM. Using sFLC measurement, we showed a higher response rate in patients treated on 2 prospective clinical trials than using M-spike measurement. More importantly, we demonstrated that responses occurred earlier using sFLC compared to M-spike measurement. sFLC measurement could predict response to therapy within the first month of therapy.

IgA and IgG hypogammaglobulinemia are a common finding in patients with WM which may contribute to recurrent infections seen in up to half of WM patients. The etiology for this finding remains unclear. As part of these studies, we investigated the prevalence and genetic basis for IgA and IgG hypogammaglobulinemia among 89 previously untreated patients with the clinicopathological diagnosis of WM. Their median IgM was 2,980 (180–12,400) mg/dl, median BM involvement was 35% (5%–95%) and median B2M was 2.7 (1.4–13.7 mg/dL). Fifty-six (63%) and 66 (74%) of these patients demonstrated IgG (

Background: Multiple myeloma (MM) is characterized by widespread involvement of the bone marrow (BM) at diagnosis, implying a continuous (re) circulation of the MM cells in the peripheral blood and (re) entrance into the BM. The normal process of B cell homing is regulated by cytokines and receptors such as SDF-1, CXCR4, VLA4, LFA-1, VCAM-1 and ICAM-1. In order to better understand the role of homing in MM, we developed an in vivo model which allows the continuous real-time imaging of MM cells as they home and adhere to the BM, as well as quantifying the numbers of cells in the circulation. Methods: MM.1S (2 ×106/ml) were fluorescently labeled by incubation with the dialkylcarbocyanine membrane dye “DiD” (Molecular Probes) 1uM dye for 15 min at 37°C. Cells were i.v injected in Balb/c mice. Appropriate arterioles in the ear pinnae of the mice were chosen for obtaining measurements, and the fluorescence signal on the MM cells was excited as the labeled cells passed through a slit of light (from a 632 nm He:Ne laser) focused across the vessel. Cell counts were obtained every 5 min from the time of injection. MM cell homing to bone marrow vasculature of the skull was analyzed using fluorescence confocal microscopy. A small incision was made in the scalp so as to expose the underlying dorsal skull surface. The mouse was placed on a warmed microscope stage. Imaging duration was 1–3 hours per session. DiD was excited with a 635 nm diode laser. High-resolution images with cellular details were obtained through the intact mouse skull at depths of up to 250 um from the surface of the skull. Images from several depths were obtained and z-stacking was performed to merge the images. Quantitative evaluation was made by dividing the bone marrow into pre-determined quadrants (areas 1 to 4) and counting numbers of fluorescent cells per field. To demonstrate that that this new model identifies changes in homing of MM cells, we used the CXCR4 inhibitor AMD3100 and anti-VLA-4 antibody to inhibit homing to the BM. MM cells were pre-incubated with AMD3100 (50 uM overnight, Sigma, MO) or anti-VLA-4 antibody (1hr incubation with 10 ug/ml, BD Pharmingen, CA) or control PBS under the same conditions. In vivo flow cytometry and confocal imaging were then performed on control and AMD3100 treated mice. Results: The number of cells in the control group decreased dramatically (86% decrease) after 1 hour indicating homing, whereas there was only a 47% reduction in the cells at 1 hour in the AMD3100 treated cohort, (p=0.002). Similarly, we demonstrated that the number of cells present in the perivascular bone marrow niches of the skull was significantly higher in the control mice as compared to the AMD3100-treated group at 1 hour after injection. The mean cell count in the AMD3100 treated mice decreased to 38% as compared to controls, p=0.01 Likewise, the use of anti-VLA-4 antibody demonstrated significant retention of MM cells in the circulation 1 hr after injection (4% reduction in circulating cell count vs 82% for control). Conclusion: We describe a new model that detects in vivo real-time homing of MM cells from the peripheral circulation into BM niches, which can be used to study the trafficking of MM cells into and out of the BM, as well as the effect of novel agents on this dynamic process.

Background: The PI3K and NFk-B/proteasome pathways are major regulators of survival in Multiple Myeloma (MM). Previous studies have demonstrated clinical efficacy of bortezomib in MM; however, not all patients responded to this agent. mTOR inhibitors have demonstrated significant in vitro and in vivo activity in MM, specifically clinical trials with the mTOR inhibitor CCI-779 (Wyeth) in MM. Therefore, we examined whether inhibition of the PI3K pathway by mTOR and proteasome inhibitors may lead to synergistic activity in MM. Methods: MM cell lines (MM.1S, RPMI, U266, OPM2) were treated with rapamycin 1–5nM (Sigma Aldrich), bortezomib 2.5–10nM (Millenium, MA), or the combination. Cytotoxicity was measured by the MTT assay at 48 hrs; DNA synthesis was measured using thymidine uptake assay; apoptosis was studied using Apo2.7 by flow cytometry, and cell cycle regulation was determined using flow cytometry. To determine whether these agents can overcome the growth advantage conferred by bone marrow stromal cells (BMSCs), we co-cultured cell lines with stromal cells. Normal peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers. Determination of the additive or synergistic effect of the combination was calculated using the CalcuSyn software (Biosoft, MO) based on the Chou-Talalay method, with synergistic activity determined as a combination index (CI) of Results: Rapamycin induced dose-dependent cytotoxicity from 0.1nM to 1nM, with an IC50 of 5nM in MM.1S and OPM2. Interestingly, higher doses did not induce further cytotoxicity, confirming that low doses of rapamycin are as effective as higher doses. RPMI and U266 MM cell lines were less sensitive to rapamycin, with 5nM inducing 40% and 20% decrease in survival, respectively. Bortezomib induced significant inhibition of survival in all MM cell lines with an IC50 of 2.5nM, as previously reported. The combination of agents induced significant inhibition of proliferation as compared to each agent alone, specifically with the combination of 5nM rapamycin with 5nM of bortezomib. In the DNA synthesis assay, the combination of bortezomib and rapamycin was significantly cytotoxic compared to each agent alone, specifically at the dose of 5nM rapamycin and bortezomib 2.5nM. The combination of rapamycin 1 to 5 nM and bortezomib 5 to 10 nM were synergistic with a CI index less than 1.0, as in RPMI (CI=0.4) and U266 (CI=0.2) cell lines. The combination of rapamycin and bortezomib at serial concentrations did not trigger cytotoxicity in PBMCs from normal volunteers, indicating significant cytotoxicity in malignant cells, with lack of toxicity in normal PBMCs and suggesting a therapeutic index. The combination of bortezomib and rapamycin demonstrated a significant inhibitory effect on the growth of MM cell lines even in coculture with stromal cells. Cell cycle analysis demonstrated G1 arrest at 24 and 48 hrs in MM.1S cells. Similar results were obtained using primary CD138+ myeloma cells from patients. Conclusion: The combination of rapamycin and bortezomib resulted in synergistic in vitro cytotoxicity in MM cells. These results provide the framework for clinical trials evaluating the combination of CCI-779 and bortezomib in MM.

Background: Waldenstrom Macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow (BM), indicating continuous entry and egression of malignant cells in and out of the BM. The process of homing and trafficking involves chemokines, cytokines, and adhesion molecules. In this study, we sought to determine the level of chemokines, cytokines and adhesion molecules in the BM and peripheral blood (PB) of patients with WM. Method: We compared BM supernatant to serum samples from 10 patients with WM, including 5 matched samples from the same patients, and PB serum and BM supernatant from 2 normal healthy controls. The Luminex® xMAP® Multiplex (Upstate, VA) allows detection of human cytokines, chemokines, and growth factors using the Beadlite® products (Upstate, VA). We focused on chemokines that regulate migration: Eotaxin, GRO, IP10, MIP1a, MIP1b, MCP1, MCP3, MDC/CCL22, and RANTES; cytokines that regulate proliferation: Interleukin (IL)-2, -4, -6, -8, -10, and -15; and adhesion molecules: ICAM, VCAM. The means fluorescence intensity (MFI) was measured for all molecules. Chemokine and adhesion receptor expression on primary WM cells and WM cell line BCWM.1 was assessed using flow cytometry. Adhesion was measured using the in vitro adhesion assay (EMD Biosciences, CA), and the anti-VLA-4 antibody (10–100ug/ml, BD Pharmingen, CA). Results: We first compared the BM supernatant of patients with WM to healthy donors. Adhesion molecules ICAM (mean MFI 5624 in WM vs 720 in control) and VCAM (mean MFI 9096 in WM vs 2135 in control) were significantly higher in the WM supernatant, p=0.03 and p=0.05 respectively. In addition, the chemokines IP-10 (Mean MFI 8186 in WM vs 1647 in control), MDC/CCL22 (mean MFI 13015 in WM vs 4221 in control), and GRO (mean MFI 7463 in WM vs 1337 in control), that regulate migration, were significantly upregulated in WM BM supernatant. We found higher expression of MIP1a in the PB of WM patients compared to patient BM supernatant (mean MFI 5715 in PB vs 1484 in BM), p=0.02. Similarly, RANTES and IL-10 were significantly higher in patients’ PB sera. There was no statistically significant difference between the PB sera of patients and healthy controls. The expression of CXCR3 (receptor of IP-10) and CCR4 (receptor of MDC/CCL22) were highly expressed on BCWM.1 (mean 90% expression). We then determined the expression of adhesion receptors on BM CD19+ cells from patient samples and BCWM.1. The WM cells and cell line expressed very high levels of surface adhesion receptors VLA4 and LFA1 (mean expression 95%). Anti-VLA-4 antibody 10ug/ml completely abrogated adhesion to fibronectin, 6% compared to control. Conclusion: Adhesion molecules VCAM and ICAM and their receptors VLA-4 and LFA-1 were significantly elevated in the bone marrow of WM patients, indicating activation of adhesion receptors and localizing WM cells in the BM. The anti-VLA-4 antibody completely abrogated adhesion in vitro. In addition, the chemokines IP-10, MDC/CCL22 and GRO were upregulated in the BM of patients, which induce homing of cells into the BM. This study, therefore, systematically defines chemokines, cytokines and adhesion markers in WM, providing the preclinical framework for inhibition of adhesion molecules in clinical protocols to improve patient outcome in WM. * HN and XL are co-first authors.

Background: Waldenstrom’s Macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma with a median overall survival of 5 to 6 years, and most symptomatic patients succumb to disease progression. Current therapies used in upfront or relapsed settings include the monoclonal antibody rituximab, alkylator agents (e.g. chlorambucil), and nucleoside analogues (cladribine or fludarabine). With those agents, the overall response rates (ORR) ranges from 30 to 70%. The proteasome inhibitor bortezomib has recently demonstrated about 50% ORR in patients with relapsed WM. Therefore, new therapies are needed in WM to enhance the activity of current therapeutic regimens. Perifosine (NSC 639966; Keryx Biopharmaceuticals, NY) is a novel Akt inhibitor that demonstrated significant activity in vitro and in vivo in WM. Here, we examined the effect of combinations of perifosine with other conventional therapies for WM. Methods: chlorambucil (Sigma Aldricht), dexamethasone (Sigma Aldricht), doxorubicin (Sigma Aldricht), fludarabine (Berlex), rituximab (Genentech), bortezomib (Millenium), and melphalan (Sigma Aldricht) were tested alone or in combination with perifosine in WM cell lines (BCWM.1 and WM-WSU). Cytotoxicity was measured using the MTT growth inhibition assay. ADCC was measured by calcein-AM release on a Wallack Spectrafluor (485 nm excitation/535 nm emission) in the presence or absence of effector mononuclear cells for 4hrs (pretreated 24hrs with IL-2 100UI/mL, ratio effector:target (E:T) cells 40:1). Determination of the additive or synergistic effect of the combination was calculated using the CalcuSyn software (Biosoft, MO) based on the Chou-Talalay method, with a combination index (CI) Results: Perifosine induced significant cytotoxicity, with an IC50 of 5–20uM. The following doses of single agent bortezomib and cytotoxic agents induced 50% growth inhibition (IC50): bortezomib 15–20nM, fludarabine 5ug/mL, doxorubicin 0.5–1nM, and melphalan 6–10uM. Dexamethasone 100nM and chlorambucil 100uM inhibited cell growth by 20%. Rituximab (10ug/mL, 1 hr) induced 121% specific lysis by ADCC in presence of effector cells. We then studied low doses of perifosine (5uM) in combination with these drugs. Perifosine 5uM induced 20% cytotoxicity at 48hrs, that was increased to 72% with fludarabine 5ug/mL (CI=0.521), to 60% with bortezomib 10nM (CI=0.49), to 67% with doxorubicin 0.1nM (CI=0.765), to 41% with dexamethasone 100nM (CI=0.335), to 50% with melphalan 5uM (CI=0.359), and to 42% with chlorambucil 50uM (CI=0.477). Finally, perifosine 10uM in combination with rituximab (10ug/mL, 1 hour) induces 193% specific lysis by ADCC mechanism in the presence of effector cells (p Conclusion: The combination of perifosine with bortezomib, rituximab and other conventional agents used in the therapy of WM has synergistic activity. Those results provide the framework for clinical evaluation of combination of perifosine with other therapies in WM. XL and GO are co-first authors. Supported in part by an ASH Scholar Award.

Background: WM is an incurable low-grade lymphoplasmacytic lymphoma with limited options of therapy. Proteasome inhibition has been shown to induce components of the proapoptotic/terminal unfolded protein response (UPR), a signaling pathway activated by accumulation of misfolded proteins within the endoplasmic reticulum (ER). We previously found that UPR gene expression is related to disease activity in WM with a particular role for GRP78/Bip as a prognostic factor. We therefore examined tunicamycin (Sigma, St Louis, MO), a potent inducer of ER stress, for potential anti-tumor effects in WM. Methods: WM cell lines (BCWM.1 and WSU-WM), IgM secreting low-grade lymphoma cell lines (MEC1, RL) and primary CD19+ selected LPC cells from WM patients were incubated with tunicamycin (0.01–10 uM) for 24–72 hours and evaluated by MTT, thymidine uptake, and Apo2.7/PI staining for effects on proliferation and survival. Since bone marrow stromal cells (BMSC) confer growth and resistance to conventional treatments, we also tested the effect of tunicamycin on WM cells co-cultured with BMSC. Immunoblotting for caspases was also performed and expression of UPR genes determined using relative quantitative RT-PCR reaction following (0.5–16 hrs) culture with tunicamycin. Results: WM cells inherently expressed the ER chaperones GRP78/Bip and GRP94/gp96. Tunicamycin rapidly induced components of the proapoptotic/terminal UPR, including PERK, the ER stress-specific eIF-2alpha kinase; ATF6, an ER stress-induced transcription factor; and its proapoptotic target, CHOP/GADD153. Tunicamycin also induced significant cytotoxicity, and inhibited DNA synthesis with an IC50 of 0.5–1 ug/mL in all cell lines, as well as primary LPC from 3/3 WM patients. Furthermore, tunicamycin induced apoptosis in WM cells, with an increase in the sub-G1 population notable at 12 hrs. Tunicamycin induced apoptosis was preceded by caspase-12 cleavage, followed then by caspase-8, -9 and PARP cleavage. Importantly, co-culture of WM cells with the survival factors IL-6, IGF-1 as well as BMSC did not inhibit tunicamycin induced cytotoxicity. Lastly, tunicamycin did not induce cytotoxicity in healthy donor peripheral blood mononuclear or hematopoietic stem cells. Conclusion: These pre-clinical studies provide a framework for further evaluation of ER stress inducing agents as therapeutic agents in WM.

Background: Waldenström’s Macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma with as yet unknown genetic basis for its pathogenesis. Several TNF family members (CD40L, APRIL and BAFF/BLYS) are known to regulate WM growth and survival. TRAFs are a novel family of adapter proteins that facilitate pro-apoptotic (TACI) or pro-survival/differentiation (CD40, BAFFR, BCMA) receptor signaling mediated by TNF family ligands. Therefore, understanding the TRAF system in WM may yield important clues about WM growth and survival. Methods: WM cell lines (BCWM.1 and WSU-WM), IgM secreting low-grade lymphoma cell lines (MEK1, RL, Namalwa), and primary bone marrow CD19+ selected lymphoplasmacytic cells (LPC) from 20 WM patients and 6 healthy donors were evaluated for TRAF (TRAF 2, 3, 5, 6) expression using semi quantitative RT-PCR and/or western blot analysis. Results: The TNF familiy receptors CD40, BAFFR, BCMA, and TACI were expressed in all cell lines tested as well as in CD19+ selected LPC from WM patients and healthy donors. Moreover, TRAF 2, 3, 5, 6 were expressed in all cell lines by both RT-PCR and western blot analysis. In contrast, we observed loss or abnormally low expression of both TRAF 2 and 5 in 6/20 (30%) patients, whilst TRAF 3 was absent or abnormally low in 3/30 (15%) patients. TRAF 6 was expressed in all patients. Among healthy donors, we observed expression of all TRAF adapter proteins. Conclusion: Up to one third of WM patients demonstrate loss of TRAF 2 and 5 adapter proteins which facilitate signaling through the pro-apoptotic receptor TACI. Ongoing studies including gene sequencing and siRNA knockdown models are delineating a role for TRAF loss in the pathogenesis of WM.

Background: Waldenstrom’s Macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma. Recently, Bortezomib has demonstrated clinical activity in patients with relapsed WM. The purpose of this study was to investigate the in vitro effect of the new proteasome inhibitor NPI-0052 (Nereus Pharmaceuticals, CA), alone and in combination with Bortezomib, in WM. Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEK1, RL, Namalwa) were used. Bone marrow primary CD19+ malignant cells were obtained from WM patients after informed consent. Inhibition of growth was measured using MTT assays. DNA synthesis was measured using the thymidine incorporation assay. Apoptosis was determined using Apo2.7 staining, followed by flow cytometric analysis. Determination of the additive or synergistic effect of the combination was calculated using the CalcuSyn software (Biosoft, MO) based on the Chou-Talalay method with the combination index (CI) Results: NPI-0052 induced cytotoxicity and inhibition of DNA synthesis, with an IC50 of 20–30nM in all cell lines tested at 24 hrs. NPI-0052 induces 50% apoptosis at 48 hrs. Similar effects were demonstrated in primary CD19+ WM cells. Bortezomib induced cytotoxicity and inhibition of DNA synthesis with an IC50 of 20–30nM in all cell lines tested. Importantly, the combination of NPI-0052 and Bortezomib induced significant inhibition of proliferation compared to each agent alone, specifically at the combination of 2.5nM and 5nM of Bortezomib and 10nM of NPI-0052. For example, Bortezomib single agent at 2.5nM and 5 nM induced 8% and 13% cytotoxicity in BCWM.1 in vitro, respectively, while the combination Bortezomib and NPI-0052 10nM induced a synergistic cytotoxic effect, 44% (CI=0.7) and 58% (CI=0.47), respectively. The combination effect of NPI-0052 20 nM with Bortezomib 2.5nM and 5nM was also synergistic, with 61% (CI=0.7) and 66% (CI=0.7) cytotoxicity, respectively. Similar effects were observed in 3 primary CD19+ WM cells obtained from patients’ bone marrow. Conclusion: NPI-0052 has significant anti-tumor activity against WM in vitro, especially in combination with Bortezomib. These results provide the framework to further study the potential therapeutic role of NPI-0052 in WM. *XJ and XL are co-first authors.

Background: Waldenstrom Macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow (BM), and lymphadenopathy in 20% of the patients, implying continuous trafficking of WM cells into and out of the BM and lymph nodes. The normal process of B-cell homing is regulated by cytokines, chemokines, and adhesion molecules. One of the most extensively studied chemokines in migration is stromal derived factor SDF-1 and its receptor CXCR4. Here we study the role of chemokine receptors, and the SDF-1/CXCR4 axis on migration and adhesion in WM. Methods: Flow cytometry for CXC and CC chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR2, CCR4, CCR5, CCR6 and CCR7), and adhesion molecules (VLA-4 and LFA-1) on WM cell lines (BCWM.1 and WM-WSU) and patient samples was performed. Migration was determined using the transwell migration assay (Costar, NY). Cells were placed in the upper chambers of the migration assay with 1% FCS medium in the presence of serial concentrations of SDF-1 in the lower chambers. After 4 hours of incubation, cells that migrated to the lower chambers were counted. Similarly, adhesion was determined using an adhesion assay (EMD Biosciences, San Diego, CA) with 96-well plated coated with fibronectin. Immunoblotting for proteins downstream of CXCR4 was performed. The CXCR4 inhibitor AMD3100 (10–100uM, Sigma, MO) and Gi protein inhibitor pertussis toxin PTX (10–200ng/ml, Sigma, MO) were used to inhibit CXCR4 signaling. Results: The following chemokine receptors were expressed on patient CD19+WM cells with over 30% expression: CXCR1 (mean 60%), CXCR2 (mean 47%), CXCR4 (mean 47%), CXCR5 (mean 69%), CCR4 (mean 54%) and CCR6 (mean 61%). Similar expression was observed on WM cell lines. We next determined the effect of SDF-1 on migration and signaling pathways in WM. SDF-1 (10–100nM) induced migration in a bell-shaped curve with 30nM inducing maximum migration (110% compared to control). SDF-1 30nM induced a rapid activation of signaling pathways downstream of CXCR4 including pERK1/2, pAKT, and pPKC at 1 min, with maximum activation at 5min. The CXCR4 inhibitor AMD3100 inhibited migration of BCWM.1 in the presence of 30nM SDF-1, with AMD3100 10uM inhibiting migration at 59% of control, and 20 to 50uM leading to a plateau in inhibition of migration at 54% of control. AMD3100 inhibited pERK and pPKC activation, downstream of CXCR4 in a dose-dependent fashion. Similar results were observed using PTX, with inhibition of migration of WM cells at 50% compared to control. To determine the role of SDF-1 on adhesion, we first demonstrated that WM cells from patients and cell lines expressed high levels of surface VLA-4 expression (mean 95% surface expression). WM cells had an increase in adhesion to fibronectin (VLA-4 ligand) compared to BSA control. AMD3100 10uM inhibited adhesion to fibronectin (63 % of control), indicating that the SDF-1/CXCR4 axis regulates adhesion. Conclusion: CXCR4 is highly expressed on WM cells and regulates migration and adhesion, indicating a potential role in regulating WM trafficking into the BM and lymph nodes. These studies provide the preclinical framework to study CXCR4 inhibitors in the regulation of homing and adhesion in WM.

Background: We have previously demonstrated activity of the new Akt inhibitor perifosine (NSC 639966; Keryx, NY) in Waldenstrom’s Macroglobulinemia (WM). Perifosine induced complete inhibition of Akt phosphorylation along with induction of apoptosis in WM cells. However, MAPK pathways and PKC proteins were activated in response to perifosine. MAPK and PKC pathways are known to promote cell proliferation. Therefore, understanding the mechanism by which perifosine induces MEK/ERK and PKC activation is important to better understand the mechanisms of response/resistance to this novel agent in ongoing clinical trials. Methods: WM cell lines (BCWM.1, WM-WSU) were treated with perifosine or with the specific Akt inhibitor Triciribine (Biomol, PA). In addition, knockdown of Akt was performed using shRNA silencing techniques (lentivirus shRNA vector, Invitrogen, Ca). The following inhibitors were also used: PI3K inhibitor LY294002 (Calbiochem, CA) and MEK inhibitor (U0126, Calbiochem, CA). Inhibition of proliferation was measured using the MTT assay. Immunoblotting was performed at different time points. Results: Perifosine induced cytotoxicity in WM cells and induced MEK/ERK activation and pPKC activation in a dose and time dependent fashion. We then treated WM cells with perifosine in the presence or absence of the MEK inhibitor U0126 and demonstrated that the combination of the two agents induced significant synergistic activity. We sought to identify the molecular mechanism by which perifosine induces MEK/ERK activation. We demonstrated that the specific AKT inhibitor Triciribine inhibited AKT and induced cytotoxicity in WM cells in a similar fashion to perifosine. However, unlike perifosine, it did not enhance MEK/ERK activity. Similarly, using Akt shRNA, we demonstrated that, despite inhibition of Akt activation, MEK/ERK was not activated. These data indicate that the effect of perifosine on MEK/ERK pathway is not through a compensatory feedback mechanism of Akt inhibition as previously thought. Therefore, we hypothesized that the effect of perifosine on the MEK/ERK pathway is through modulation of upstream pathways, specifically PI3K, PKC and c-Raf/MEK pathways. We first demonstrated that the specific PI3K inhibitor LY294002 (25mM for 15 minutes) completely abrogated Akt phosphorylation, while inducing significant ERK activation, indicating that the effect of perifosine on MEK/ERK may be similar to that of LY294002. We also demonstrated that perifosine and LY294002 activated c-Raf and pan-pPKC at 4 hrs. Conclusion: Based on this, we believe that in the presence of perifosine, growth receptor stimulation leads to PLC and RTK activation, which induces PIP2 stimulation. PIP2 is upstream of PI3K and PKC. Given that PI3K is blocked by perifosine, PIP2 leads to activation of PKC, which then induces growth stimulation, and activation of c-Raf and downstream MEK/ERK. In addition, growth receptors may also activate Raf through the Ras/Raf/MEK pathway, independent of PKC. These studies provide a better understanding of molecular mechanisms that regulate resistance to perifosine. Future combinations of perifosine with MEK inhibitors or PKC inhibitors such as AZD6244 and Enzastaurin may overcome this resistance and induce significant activity in WM.

Introduction: WM is a B-cell disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells (LPC), along with excess mast cells (MC) which support the growth and survival of BM LPC through multiple TNF-family ligands including CD40L, APRIL and BLyS/BAFF. Importantly, BM LPC stimulate cell surface expression of TNF-family ligands through release of sCD27 which induces CD70 on MC. We therefore have sought the development of agents which could target CD27-CD70 interactions. As such, we examined the therapeutic potential of directly targeting CD70 using the fully humanized monoclonal antibody SGN-70 (Seattle Genetics, Inc., Bothell WA). Methods-Results: As part of these studies, we used flow cytometric analysis to evaluate the expression of CD70 on primary WM patient BM LPC and MC, as well as 2 WM cell lines (BCWM.1 and WM-WSU). These studies demonstrated cell surface expression of CD70 on BM LPC and MC from 20/26 (77%) and 10/11 (90%) WM patients, respectively. We next assessed the ability of the SGN-70 antibody to eradicate primary WM LPC (n=5) and WM cell lines by assessing for direct induction of apoptosis, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) as well as induction of TNF family ligands on primary WM MC and the LAD2 MC line. Following incubation of WM LPC with SGN-70 (0.01–20 μg/ml), no direct induction of apoptosis or CDC activity was observed. However, SGN-70 mediated significant dose-dependent ADCC against WM LPC and MC at concentrations of 0.1–20 ug/ml. Importantly, SGN-70 blocked sCD27-induced expression of CD40L and APRIL on primary WM MC and LAD2 MC. To further evaluate the therapeutic potential of SGN-70 in an in vivo model, SCID-hu mice bearing BCWM.1 WM cells were treated with SGN-70 (1 mg/kg, i.p., qOD) Serum human IgM and sCD27 levels were measured by ELISA to monitor for tumor engraftment and disease progression. SGN-70 initiated 6 weeks following tumor engraftment blocked tumor growth in 12/12 treated mice, whereas all 5 untreated mice demonstrated disease progression. The results of these studies provide the framework for clinical trials to examine the therapeutic potential of the SGN-70 monoclonal antibody in WM.

Background: Waldenstrom’s Macroglobulinemia (WM) is a low-grade lymphoplasmacytic lymphoma with limited options of therapy. The PI3k/Akt pathway is a critical regulator of cell survival. Our previous studies using proteomic analysis have demonstrated upregulation of members of the PI3k/Akt pathway in WM. We examined whether the new Akt inhibitor perifosine (NSC 639966; Keryx, NY) induces cytotoxicity in WM. Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Primary CD19+ malignant cells were obtained from patients after informed consent. Inhibition of proliferation was measured using the MTT assay; DNA synthesis was measured using the thymidine uptake assay and apoptosis using Apo2.7 flow cytometry. Bone marrow stromal cells (BMSC) confer growth and resistance to conventional treatments. We therefore, tested the effect of perifosine on WM cells co-cultured with BMSC. Immunoblotting for signaling pathways was performed at different time (30 minutes to 16 hrs) and doses of therapy. In vivo activity of perifosine was assessed using a SCID-irradiated model with subcutaneous tumors in which perifosine was administered by oral gavage daily (35 mg/kg/day). A two-sided t-test was used to determine statistical differences. Results: Perifosine inhibited phosphorylation of Akt in a dose- and time- dependent fashion, as well as downstream GSK3a/b and ribosomal phospho-S6. Perifosine inhibited Akt activity as confirmed by Akt kinase assay. Perifosine induced significant cytotoxicity and inhibition of DNA synthesis with an IC50 of 5-20uM in all cell lines tested. Similar effects were observed in primary CD19+ patient WM cells. Perifosine induced apoptosis in WM cells as demonstrated by flow cytometry. The mechanism of apoptosis induced by perifosine was through activation of SAPK/JNK pathway, followed by caspase-8, -9 and PARP cleavage. The JNK inhibitor SP600125 abrogated perifosine-induced apoptosis. The growth inhibitory effects of perifosine were significant even in the presence of BMSC, IL-6 and IGF-1, which induce resistance to conventional therapies. Importantly, perifosine did not induce cytotoxicity in healthy donor peripheral blood mononuclear cells or in hematopietic stem cells in a methylcellulose colony forming cell assay, indicating lack of toxicity on normal cells. Interestingly, MAPK members such as MEK/ERK were activated by perifosine. The MEK inhibitor U0126 significantly enhanced perifosine-induced cytotoxicity in WM cells, indicating that this combination may be synergistic in vivo. Finally, perifosine induced significant reduction in WM tumor growth in vivo, as compared to control cohort treated with vehicle only at week 6 (p=0.05). Conclusion: Perifosine has significant antitumor activity in WM both in vitro and in vivo. These results provide the framework for clinical evaluation of perifosine in WM. Supported in part by the Leukemia and Lymphoma Society, the Lymphoma Research Foundation and an American Society of Hematology Scholar Award. * XL and XJ are co-first authors.

Background: Waldenstrom’s Macroglobulinemia is an incurable lymphoplasmacytic lymphoma with limited options of therapy. We have previously demonstrated upregulation of PKCβ protein in WM using protein array techniques, and confirmed increased expression in WM using immunohistochemistry. PKCβ regulates cell survival and growth, as well as migration and homing in many B-cell malignancies. We therefore hypothesized that inhibition of PKCβ will induce cytotoxicity in WM. Methods: In this study, we examined the effect of serial dilutions of the PKCβ enzastaurin (2.5 uM to 20 uM) on WM cell lines (BCWM1 and WM-WSU), IgM secreting low-grade lymphoma cell lines (MEC-1, RL), as well as primary CD19+ WM cells and WM cells adherent to bone marrow stromal cells (BMSCs), which induce resistance to conventional therapy. Cytotoxicity was measured by MTT assay and inhibition of cell proliferation was determined by thymidine uptake assay. Apoptosis was measured by flow cytometry using Annexin V and DAPI staining at 48 h. Cell DNA content analysis was performed using DAPI staining on fresh cells. Cell signaling pathways targeted by enzastaurin were determined using immunoblotting at 6 h (2.5 to 10 uM) and at 7.5 uM (10 min to 12 h). The effect of enzastaurin in vivo was determined using a subcutaneous WM model in SCID mice. Enzastaurin was given by oral gavage (80mg/kg twice daily). Results: Enzastaurin demonstrated time and dose-dependent inhibition of PKCβ in WM cells. It induced a significant decrease of proliferation at 24 and 48 h in all cell lines tested with an IC50 of 2.5 to 10 uM, even in the presence of DOPPA, a specific PKCβ stimulator. Similar effects were demonstrated in primary CD19+ WM cells, with no cytotoxicity on peripheral blood mononuclear cells indicating selective toxicity on malignant cells. Enzastaurin induced dose-dependent apoptosis at 24 and 48 h with induction of caspases 3, 8, 9 and PARP cleavage as well as a decrease in Bcl-xL. Analysis of cell DNA content confirmed apoptosis at low doses of enzastaurin (5 uM). To further determine the mechanism of action of enzastaurin in WM, we examined downstream molecules. It significantly inhibited AKT phosphorylation and AKT kinase activity, as determined by inhibition of phosphorylation of GSKα/β fusion protein. In addition, enzastaurin inhibited p-MARCK, and ribosomal p-S6. Enzastaurin overcame resistance induced by co-culture of WM cells with bone marrow stromal cells. In addition, enzastaurin (2.5 to 5 uM) in combination with bortezomib (2.5 to 10 nM), another active agent in WM, demonstrated strong synergistic activity using the Calcusyn software for synergy. Given that PKCβ regulates migration and homing of B-cells, we next determined the effect of enzastaurin on in vitro migration of WM cells. In the transwell migration assay, enzastaurin inhibited migration in a dose-dependent fashion (p=0.041). Finally, in vivo animal studies demonstrated significant inhibition of WM tumor growth in the enzastaurin treated mice (n=11), compared to control mice (n=8) (p=0.028). Conclusion: Enzastaurin has significant antitumor activity in WM in vitro and in vivo, providing the framework for clinical trials evaluating enzastaurin as a new therapeutic agent in patients with WM.