Absence Of Lenalidomide Research Articles

Exhausted T Cells Characterized By Upregulation of Specific Transcription Factors Are Increased in a Mouse Model of De Novo Mature B Cell Neoplasms

Introduction: Recent studies demonstrated that exhausted T cells expressing immune checkpoint molecules such as PD-1, Tim-3, and TIGIT are increased at tumor sites in diffuse large B cell lymphoma (DLBCL) and multiple myeloma (MM) patients. However, the pathogenetic significance of exhausted T cells remains controversial and needs thorough investigation. Method: In this study, we used a mouse model with human MM mutations of conditional Utx loss and BrafV600E expression only in mature B cells, which developed mature B cell neoplasms de novo with time. Therefore, we expected this mouse model to show immune escape similar to human patients, rather than the currently available transplanted mouse models. To examine the level of T cell exhaustion, we analyzed the expression of PD-1 and Tim-3 on CD8+ T cells. To understand the characteristics of exhausted T cells, we performed flow cytometry (FCM) and RNA-seq analyses. Result: In mice with mature B cell neoplasms, PD-1+ Tim-3+ and PD-1+ Tim-3- T cells were significantly increased in BM, spleen, and lymph nodes, compared with control mice (p<0.0001). The expression level of TIGIT, which was reported to be expressed on CD8+ T cells of MM patients more frequently than other inhibitory receptors, was significantly higher in PD-1+ Tim-3+ T cells. These data suggest that the increase in PD-1+ Tim-3+ and PD-1+ Tim-3- T cells at the tumor site may be related to the tumor pathogenesis in our mouse model. We then investigated the functional and transcriptional features of these exhausted T cells. The FCM analyses revealed that PD-1+ Tim-3+ T cells had the highest production capacity of granzyme B and expressed the highest level of Ki-67 and annexin V, compared with PD-1+ Tim-3- and PD-1- Tim-3- T cells. These characteristics are consistent with those of terminally exhausted T cells previously reported. RNA-seq analyses also revealed the similarities between PD-1+ Tim-3+ T cells in this mouse model and terminally exhausted T cells. The transcription factors Tox and Nr4a2, which reportedly contribute to the progression of T cell exhaustion, were up-regulated in PD-1+ Tim-3+ T cells. On the other hand, PD-1+ Tim-3- T cells showed high production capacity of IFN-γ and TNF-α and expressed moderate levels of Ki-67 with less apoptotic features. RNA-seq demonstrated the enrichment of gene sets related to immune system process. Tox and Nr4a2 were also up-regulated in PD-1+ Tim-3- T cells but their levels were lower than in PD-1+ Tim-3+ T cells. Furthermore, PD-1+ Tim-3- T cells retained the expression of Tcf7, encoding TCF-1 which plays a significant role in maintaining stem-like features. These results indicate that PD-1+ Tim-3+ T cells have direct cytotoxicity and strong proliferative capacity, however most of these cells are severely apoptotic. Therefore, PD-1+ Tim-3+ T cells are possibly unable to keep long-term anti-tumor functions. In contrast, PD-1+ Tim-3- T cells show immunological responsiveness and progenitor-like transcriptional features, suggesting that these cells may maintain effective immune functions for a long time. When we stimulated CD8+ T cells in vitro in the presence or absence of lenalidomide or pomalidomide, these IMiDs down-regulated the expression of Tox and Nr4a2, suggesting that they might prevent T cell exhaustion. Conclusion: The present study using our mouse model revealed the heterogeneity and the increase in exhausted T cells during the development of de novo mature B cell neoplasms. PD-1+ Tim-3+ T cells exhibit terminally exhausted features, whereas PD-1+ Tim-3- T cells retain stem-like transcriptional features. IMiDs might prevent excessive T cell exhaustion and contribute to the immune response with long-term effects.

Lenalidomide Promotes a Pro-Inflammatory Switch of Nurse-like Cells Derived from Chronic Lymphocytic Leukemia

Lenalidomide is an immunomodulatory agent clinically active in CLL patients. The specific mechanism of action is still undefined, but includes the modulation of microenvironment. In CLL patients, nurse-like cells (NLCs) differentiate from CD14+ mononuclear cells and nurture/protect CLL cells from apoptosis. NLCs resemble M2 macrophages with potent immunosuppressive functions.We examined the ability of lenalidomide to mediate a pro-inflammatory switch of NLCs affecting the protective microenvironment generated by CLL into tissues.NLCs were generated in presence or absence of lenalidomide: cell surface markers, phagocytosis and induction of T cell proliferation were analyzed after 10 days. NLCs activation was measured using a yellow tetrazolium MTT assay after 5 days of culture and NLCs proliferation was measured by CFSE staining. Microarray-based gene expression profiles of NLCs treated or not with lenalidomide were evaluated after 10 days and data were confirmed by real time PCR.Lenalidomide modifies the immunophenotype and the biological characteristics of NLCs. First, treatment with lenalidomide 0.5µM and 1µM increased the number of NLCs to 268% and 309% compared to untreated control (100%) respectively (p<0.05). To explain the high number of NLCs generated by lenalidomide, we analyzed cell activation and proliferation. We observed a strong increase in NLCs activation after treatment with lenalidomide that correlated with stimulation of NLCs proliferation from 44% to 55% (% of dividing cells) (p<0.05).In contrast with the high number of NLCs generated in presence of lenalidomide, we found that NLCs lost the ability to nurture and protect CLL cell from apoptosis reducing their viability from 54.2% to 44.5% (p<0.05), but they strongly attracted CLL cells reaching an increase of adhesion to 227% and 212% with the addition of 0.5µM and 1µM lenalidomide (p<0.05). Accordingly with these results, we investigated the ability of lenalidomide to interfere with leukemia-promoting activity of NLCs. Lenalidomide improved the ability of NLCs to engulf zymogen particles to 141% and 155% with dosage of 0.5µM and 1µM compared to control (100%) (p<0.05), further confirmed analyzing the uptake of FITC-dextran by NLCs that increased to 252% and 356% compared to untreated control (p<0.01). Moreover lenalidomide strongly improved the ability of NLCs to induce T cells proliferation from 19.5% to 35.0% (% of dividing cells) (p<0.05). Lastly, gene expression profiling showed a switch to a pro-inflammatory profile in NLCs induced by treatment with lenalidomide involving a modulation of pivotal genes for the immune response, activation/proliferation of T cells, complement activation as well as regulation of cellular movement, cytokine and chemokine activation. In particular, down-regulation of CCL2, CXCL12, IL-10, CD163 and up-regulation of IL-2 were apparent.Collectively, our data provide new insights into the mechanism of action of lenalidomide that reverts NLCs polarization from M2 to M1-skewed phenotype affecting the supporting and protective microenvironment generated by CLL into tissues. DisclosuresMaffei:Celgene: support for travel to congresses Other. Marasca:Celgene: Honoraria, Research Funding.

Lenalidomide Induces Lipid Raft Assembly to Enhance Erythropoietin Receptor Signaling in Myelodysplastic Syndrome Progenitors

Erythropoietin receptor (EpoR) signaling is impaired in patients with Myelodysplastic Syndromes (MDS) despite appropriate growth factor production and cellular receptor display. We previously reported that EpoR signaling is dependent upon receptor localization within membrane lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity (McGraw KL, et al. PLoS One 2012). Here, we show that MDS erythroid progenitors display markedly diminished raft assembly (p=0.005) and smaller raft aggregates (p=0.023) compared to normal controls. Because lenalidomide triggers raft coalescence in T-lymphocytes to promote immune synapse formation, we assessed the effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lipid rafts were isolated from UT7 cells using ultracentrifugation and identified by GM-1 dot blot and Lyn kinase western blot. Lenalidomide rapidly induced lipid raft formation in UT7 cells which was confirmed by confocal microscopy visualization of GM-1 fluorescence. Lenalidomide also significantly induced lipid raft formation in pooled MDS erythroid progenitors (CD71+, cKit+) from 11 patients [mean raft size, control (n=569) vs. lenalidomide treatment (n=659), p<0.001], with no significant change observed in pooled erythroids from 3 normal donors (n= 327 for control and n=365 for lenalidomide treated, p=0.37). Interestingly, lipid rafts were significantly larger in erythroid progenitors from patients who responded (n=5) to lenalidomide treatment compared to non-responders (n=3) (75.52 ±13.68 vs. 35.85 ±8.56, p=0.02). Although lenalidomide increased raft size in erythroid progenitors from both responders (p=0.0007) and non-responders (p=0.013), mean raft size was greater in erythroid precursors from responding patients after treatment (p=0.11). Increased raft aggregation after lenalidomide treatment was accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase, whereas the JAK2 phosphatase, CD45, a negative regulator of EpoR signaling, was displaced from raft fractions. Incubation with lenalidomide prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 cells and primary MDS erythroid precursors. Bone marrow specimens from 12 non-del(5q) IPSS lower risk, lenalidomide naive MDS patients were analyzed by flow cytometry to compare changes in STAT5 phosphorylation in response to Epo stimulation in the presence or absence of lenalidomide. We found a 79.1% mean increase in p-STAT5 mean fluorescence intensity (MFI 95th percentile) in CD45dim, CD71high, Glylow erythroid precursors in 7 of the 12 patient specimens following lenalidomide exposure. Furthermore, increased STAT5 phosphorylation was accompanied by increased DNA binding of the transcription factor in UT7 cells, and improved erythroid colony forming capacity in both UT7 and primary MDS bone marrow cells. Raft induction was associated with F-actin polymerization that was blocked by Rho kinase inhibition and confirmed by lipid raft isolation followed by dot blot with western blot and confocal microscopy. These data provide new insight into abnormalities in the EpoR signaling platform that underlie impaired Epo responsiveness in MDS erythroid precursors. Our findings that deficient raft integrity impairs EpoR signaling provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS. These data also warrant investigation in a larger data set to determine whether lipid raft size may be a predictive biomarker for lenalidomide response. DisclosuresList:Celgene: Consultancy.

Mesenchymal stromal cells from patients with myelodyplastic syndrome display distinct functional alterations that are modulated by lenalidomide

The contribution of the bone marrow microenvironment in myelodysplastic syndrome is controversial. We therefore analyzed the functional properties of primary mesenchymal stromal cells from patients with myelodysplastic syndrome in the presence or absence of lenalidomide. Compared to healthy controls, clonality and growth were reduced across all disease stages. Furthermore, differentiation defects and particular expression of adhesion and cell surface molecules (e.g. CD166, CD29, CD146) were detected. Interestingly, the levels of stromal derived factor 1-alpha in patients' cells culture supernatants were almost 2-fold lower (P<0.01) than those in controls and this was paralleled by a reduced induction of migration of CD34(+) hematopoietic cells. Co-cultures of mesenchymal stromal cells from patients with CD34(+) cells from healthy donors resulted in reduced numbers of cobblestone area-forming cells and fewer colony-forming units. Exposure of stromal cells from patients and controls to lenalidomide led to a further reduction of stromal derived factor 1-alpha secretion and cobblestone area formation, respectively. Moreover, lenalidomide pretreatment of mesenchymal stromal cells from patients with low but not high-risk myelodysplastic syndrome was able to rescue impaired erythroid and myeloid colony formation of early hematopoietic progenitors. In conclusion, our analyses support the notion that the stromal microenvironment is involved in the pathophysiology of myelodysplastic syndrome thus representing a potential target for therapeutic interventions.

Immunomodulatory drugs lenalidomide and pomalidomide inhibit multiple myeloma-induced osteoclast formation and the RANKL/OPG ratio in the myeloma microenvironment targeting the expression of adhesion molecules

Multiple myeloma (MM)-induced osteoclast (OC) formation is mainly due to an imbalance of the receptor activator NF-κB ligand (RANKL)-osteoprotegerin (OPG) ratio in favor of RANKL in the bone microenvironment and to the CCL3 production by MM cells. The purpose of the study was to investigate the effect of the immunomodulatory drugs on RANKL/OPG ratio, the production of pro-osteoclastogenic cytokines, and MM-induced OC formation. We found that invivo concentrations of both lenalidomide (LEN) and pomalidomide (POM) significantly blunted RANKL upregulation normalizing the RANKL/OPG ratio in human osteoprogenitor cells (PreOBs) when co-cultured with MM cells and also inhibited CCL3 production by MM cells. A reduction in CD49d expression, a molecule critically involved in RANKL upregulation in the MM microenvironment, accompanied this effect. Consistently, the pro-osteoclastogenic property of MM cells co-cultured with PreOBs was reduced by both LEN and POM. We further investigated the effect of these drugs on the transcriptional profile of both MM cells and PreOBs by microarray analysis, which showed that adhesion molecules, such as ITGA8 and ICAM2, are significantly downregulated in MM cells. Our data suggest that LEN and POM inhibit MM-induced OC formation through normalization of the RANKL/OPG ratio targeting the expression of adhesion molecules by MM cells.

Lenalidomide Reduces Survival of Chronic Lymphocytic Leukemia Cells in Primary Co-Cultures by Altering the Myeloid Microenvironment

Abstract 3894Chronic lymphocytic leukemia (CLL) cells are highly dependent on their microenvironment. External stimuli provided by bone marrow stromal cells or non-malignant leukocytes are required for their survival and proliferation. Interestingly, peripheral blood-derived monocytes differentiate in the presence of CLL cells to so-called Nurse-like cells (NLCs), which are round or fibroblast-shaped adherent cells that were shown to promote survival of CLL cells in vitro and to exist in lymph nodes of CLL patients. In search of new therapeutic options for patients with CLL, the immunomodulatory drug lenalidomide turned out to have significant clinical activity in CLL. Lenalidomide does not induce apoptosis in CLL cells directly, but is rather believed to act via the microenvironment. Several studies described that it alters cytokine levels and the activation status of the cells. Further, a CLL-specific T-cell defect was shown to be repaired by lenalidomide, which might represent a major activity of this drug in CLL. However, its mechanism of action seems to be complex and is not well understood.As monocytes as well as NLCs are very effective in maintaining survival of CLL cells, we aimed to investigate whether lenalidomide interferes with these supportive cell-cell interactions. To do this, we established primary co-cultures of monocytes and CLL cells in the presence or absence of lenalidomide and observed a significantly decreased viability of CLL cells after 14 days of treatment, suggesting an impact of this drug on the survival support of NLCs. Therefore, we analyzed the immunophenotype of NLCs by flow cytometry, as well as the secretion of cytokines in the co-cultures by ELISA and antibody-coupled bead arrays. Among the effects induced by lenalidomide, we observed reduced cell surface expression of the MHC II protein HLA-DR on NLCs as well as lower levels of the chemokine CCL2, but higher levels of IL-10 in the culture supernatant, indicating an altered inflammatory milieu in the co-cultures. The enhanced IL-10 levels resulted in an increase in STAT1 phosphorylation in CLL cells as measured by Western blot analysis. As a consequence, enhanced expression of the adhesion molecule ICAM-1 (CD54) and an altered expression of cytoskeletal genes (e.g. RHOC and CORO1B) were observed in CLL cells after lenalidomide treatment. Chemotaxis assays using transwell culture dishes and SDF1-α as chemoattractant revealed an impaired migratory potential of lenalidomide-treated CLL cells, which was not due to reduced expression of the SDF1-α receptor CXCR4. In summary, our data show that lenalidomide reduces the survival support of NLCs for CLL cells in vitro, suggesting that this drug effects the myeloid microenvironment in CLL in vivo. Furthermore, lenalidomide impairs the migratory potential of CLL cells which may affect circulation and homing of CLL cells in vivo. Disclosures:No relevant conflicts of interest to declare.

The Activity of MOR202, a Fully Human Anti-CD38 Antibody, Is Complemented by ADCP and Is Synergistically Enhanced by Lenalidomide in Vitro and in Vivo

AbstractAbstract 4018 Background:MOR202, a HuCAL-derived fully human anti-CD38 antibody has proven to be highly effective in preclinical models of multiple myeloma (MM) and is currently evaluated in a phase I/II clinical trial. Having previously identified ADCC as potent effector mechanism of MOR202, the capability to induce killing of MM patient cells via antibody dependent cellular phagocytosis (ADCP) was assessed. Furthermore, the immunomodulatory agent lenalidomide (LEN), a commonly used agent in MM therapy was investigated for its potential to enhance the cytotoxicity of MOR202 in vitro and in vivo. Methods:Drug effects in presence or absence of LEN were characterized in vitro in a FACS-based phagocytosis assay using macrophages derived from M-CSF stimulated monocytes and a panel of CD38+ MM target cells. The therapeutic potential of MOR202 in vivo as a single agent and in combination with LEN was studied in a disseminated survival model of iv injected Ramos lymphoma cells. Synergy was determined using the fractional product concept. Results:MOR202 induced ADCP with high potency showing EC50 values between 40 and 130pM. Maximal killing was donor and cell line dependent ranging from 11 to 72%. The combination with LEN induced additive to synergistic enhancement of MOR202 activity which was mediated by several mechanisms identified to be direct cytotoxicity and increased CD38 expression levels on MM cells. In vivo, MOR202 at 1mg/kg increased median survival (MS) by 142% compared to vehicle control (MS day 48.5 vs. day 20), while LEN had no distinct effect at any of the tested doses between 25 and 100mg/kg (MS day 21–22). Intriguingly, despite the apparent lack of efficacy by LEN alone co-administration of 1mg/kg MOR202 and 100mg/kg LEN resulted in a superior increase of MS by 225% (MS day 65), thus demonstrating strong synergism. Of note, while no mouse survived beyond day 65 in any other group, 3/8 animals in the combo group were completely free of tumor until study termination on day 98. Conclusions:We here demonstrate ADCP to be an additional potent effector mechanism of MOR202. The combination of MOR202 and LEN enhanced ADCP-mediated killing in vitro and increased median survival in vivo in a synergistic manner. These results suggest a mechanistic rationale for MOR202 combinations with LEN including in particular LEN non-responders or relapse/refractory patients, thus warranting further evaluation in future clinical trials. Disclosures:Endell:MorphoSys AG: Employment. Boxhammer:MorphoSys AG: Employment. Wurzenberger:MorphoSys AG: Employment. Ness:MorphoSys AG: Employment. Steidl:MorphoSys AG: Employment.

Lenalidomide Modulates the Phenotype and Function of Human Mesenchymal Stromal Cells From Healthy Donors and MDS Patients.

Abstract 3816Poster Board III-752 IntroductionRecent studies in patients with MDS have clearly demonstrated the clinical efficacy of lenalidomide. However, its exact mechanisms of action have not been elucidated yet. Myelosuppression is the most common adverse event and seems to be dependent on dose as well MDS subtype, being rather infrequent in patients other than del5q. The aim of this study was to investigate whether lenalidomide affects the bone marrow microenvironment. Therefore, we analyzed in-vitro characteristics of isolated mesenchymal stromal cells (MSCs) from MDS patients and from healthy controls. MethodsBone marrow samples were collected from healthy donors (n=5) and patients with MDS (del5q MDS n=3, RA n=2, RAEB1/2 n=3). MSCs were isolated according to the standard adhesion protocol and cultured in the presence or absence of lenalidomide. ResultsLenalidomide treatment of MSCs caused no morphological changes but proliferation was slightly increased. Typical surface molecules as CD73, CD90, CD105 and CD166 were expressed in MSCs from MDS patients at comparable levels to healthy controls. Lenalidomide treatment caused an upregulation of CD29 by 17.8 ± 4.4% and of CD73 by 24 ± 5.7% (mean fluorescence intensity). Investigating the cytokine production, we found lower IL-8 mRNA and protein levels in MSCs from MDS patients (mean in MDS MSC: 138.1 pg/ml vs. mean in healthy MSC: 1177 pg/ml). Interestingly, the IL-8 production can be increased by approximately 40% under lenalidomide treatment. MDS MSCs retained the capacity for adipogenic and osteogenic differentiation as well as their supportive function towards hematopoietic cells in long term culture-initiating assays (LTC-IC). However, the LTC-IC frequency was lower on MSC which had been preincubated with lenalidomide compared to controls. Lenalidomide also slightly accelerated osteogenic differentiation because mineralization started as early as on day 5 with lenalidomide whereas in the control cells first calcium deposits were visible after 7 days. Other samples showed augmented lipid vacuoles after adipogenic differentiation under lenalidomide treatment. ConclusionIn conclusion, lenalidomide modulates the phenotype of MSC and leads to an increase of their IL-8 secretion by a yet unknown mechanism. Whether these in-vitro effects are associated with the clinical efficacy of this compound in patients with MDS remains to be investigated. Disclosures:Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Lenalidomide and Bortezomib: Targeting Osteoclastogenesis, Osteoclast Survival Factors, and Bone Remodeling Markers in Multiple Myeloma.

Osteolytic bone disease in Multiple Myeloma (MM) is caused by enhanced osteoclast (OCLs) activation and inhibition of osteoblast function. Lenalidomide and bortezomib have shown promising anti-MM effects, and bortezomib has inhibitory effects on OCLs. However, the effect of lenalidomide on OCLs in MM and how bortezomib interferes with osteoclastogenesis is unknown. Here we investigated the effect of lenalidomide and bortezomib on human OCLs. Peripheral blood mononuclear cells (PBMC) from MM patients were stimulated with receptor activator of NFk-B ligand (RANKL) (50ng/ml) and M-CSF (25ng/ml) for two weeks to induce OCL formation, in the presence or absence of lenalidomide or bortezomib. OCLs were identified by flow cytometric analysis using anti-αVβ3 integrin. Lenalidomide and bortezomib inhibited OCL differentiation indicated by a decrease in αVβ3-integrin (lenalidomide at 0μM: median 69.3%; range 28.9 – 89.0%; at 2μM: median 50.4%; range 21.5 – 64.2%; at 10μM: median 39.2%; range 33.6 – 47.5%) (bortezomib at 0nM: median 69.3%; range 28.9 – 89.0%; at 2nM: median 35.0%; range 11.0 – 79.0%; at 5nM: median 11.5%; range 5.5 – 8.8%; p<0.05). Tartrate-resistant acid phosphatase (TRAP) staining was performed to identify OCLs and confirm OCL activity. Both drugs decreased TRAP -positive cells (lenalidomide at 0μM: median 70.5%; range 50.0 – 84.0%; at 2μM: median 47.0%; range 31.0 – 77.0%; at 10μM: median 32.5%; range 14.0 – 44.0%; p<0.05) (bortezomib at 0nM: median 70.5%; range 50.0 – 84.0%; at 2nM: median 26.0%; range 11.0 – 39.0%; p<0.01; at 5nM: median 17.0%; range 13.0 – 34.0%; p<0.001). To assess bone resorption activity, OCL were cultured with dentine discs, in the presence or absence of lenalidomide and bortezomib, followed by light microscopic analysis. Both lenalidomide and bortezomib inhibited bone resorption in a dose-dependent manner. Using western blot analysis, we identified PU.1 and pERK as major targets of lenalidomide, and NFATc1 as a target of bortezomib, resulting in inhibition of osteoclastogenesis. Furthermore, downregulation of cathepsin K, essential for resorption of the bone collagen matrix, was also noted. We demonstrated a significant decrease of growth and survival factors including MIP-1α, IL-6, B-cell activation factor (BAFF), and a proliferation-inducing ligand (APRIL). Importantly, in serum from patients with refractory of relapsed MM treated with lenalidomide, the essential bone remodeling factor RANKL, as well as the RANKL/Osteoprotegerin (OPG) ratio, were significantly reduced. The median OPG at baseline was significantly lower (median 8.8pg/ml, range 7.7–12.6pg/ml) than after treatment (median 10.4pg/ml, range 8.5–15pg/ml) (p<0.05). Lenalidomide also significantly inhibited the secretion of RANKL in patient's serum (baseline: median 17pg/ml, range 9.0–36pg/ml; two months after therapy: median 4.2pg/ml, range 2.5–36pg/ml) (p<0.05). The ratio of RANKL/OPG was significantly higher before than after therapy (baseline: median 2.2pg/ml, range 1.1–3.0pg/ml; two months after therapy: median 0.5pg/ml, range 0.3–2.6pg/ml) (p<0.05). We conclude that both agents specifically target key factors in osteoclastogenesis, thereby inhibiting OCL differentiation and function.

Lenalidomide Abrogates the Protective Influence of Nurse-Like Cells on Primary Chronic Lymphocytic Leukemia Cells In Vitro.

Lenalidomide (3-(4-amino-1-oxo-3H-isoindol-2-yl)piperidine-2,6-dione)) is an agent approved for treatment of patients with del 5q myelodysplastic syndromes and previously treated multiple myeloma. Lenalidomide has been found in early clinical trials to have potential therapeutic activity in patients with relapsed chronic lymphocytic leukemia (CLL). The mechanism(s) whereby this drug is active in CLL is unknown. In particular, studies to date have not found lenalidomide to have any direct cytotoxic activity on CLL cells in vitro. This has stimulated speculation that this agent might adversely affect the positive influence of the microenvironment on leukemia-cell survival. We and others have observed that cells found in the leukemia microenvironment can support CLL-cell survival in vitro. One such type of cells are nurse-like cells (NLC), which can differentiate from the CD14-positive blood mononuclear cells of CLL patients into large, round adherent cells that can attract and support CLL cell survival in vitro for weeks, if not longer. We evaluated the effects of lenalidomide on primary leukemia-cell survival in vitro when the CLL cells from different patients (N=21) were cultured alone or together with NLC generated as previously described [Tsukada Blood 2002]. We assessed the in-vitro activity of lenalidomide on primary CLL cells from 21 patients, in duplicate in a series of 6 experiments. Lenalidomide at concentrations of 0.1μM-200μM did not significantly impact the survival of CLL cells that were cultured alone for up to 12 days. Analysis of cell surface markers revealed increased expression of CD38 at 36 hours in 5/5 lenalidomide treated CLL samples compared with untreated cells (MFIR 5.7 +/− .86 vs. 3.4 +/− .83 p=.003). We observed sustained upregualtion of CD40 and regulation of CXCR4 in the majority of cells treated with lenalidomide. When cultured with NLC, the survival of CLL cells was comparable to or significantly higher than that of CLL cells cultured alone 62.4% vs. 51% (+/−3% SEM n=21 p [<] 0.0005). The addition of lenalidomide at concentrations of 0.1μM and greater to co-cultures of NLC and CLL cells caused specific reductions in CLL cell survival to levels similar to or lower than that of CLL cells cultured without NLC. In the presence of NLC, lenalidomide at 1μM reduced CLL cell viability compared to control (41.5% vs. 56% +/−4% p [<] 0.0005 paired student t test n=13). For most patients the levels of CLL cell viability on days 4 through 8 in the co-cultures with lenalidomide was significantly lower than those of CLL cells co-cultured with NLC in the absence of lenalidomide. As such, this study reveals that physiologic concentrations of lenalidomide might abrogate the protective influence of NLC on CLL cell survival in vitro and potentially in vivo. Conceivably, those patients who have leukemia cells displaying a high dependency on NLC for survival in vitro also might be most likely to experience a favorable clinical response to treatment with lenalidomide. This hypothesis will be tested in a prospective manner with a planned clinical trial evaluating lenalidomide for treatment of CLL through the CLL Research Consortium.

Lenalidomide and Bortezomib Inhibit Osteoclast Differentiation and Activation in Multiple Myeloma: Clinical Implications.

Osteolytic bone disease in Multiple Myeloma (MM) is caused by enhanced osteoclast (OCL) activation and inhibition of osteoblast function. The proteasome inhibitor bortezomib (PS341, Velcade) has potent anti-myeloma activity with impressive clinical responses. A recent study indicated that bortezomib has inhibitory effects on OCL (ASH 2005, Abstract #2488). Lenalidomide (CC-5013, Revlimid) is an immunomodulatory derivative of thalidomide that has shown promising anti-MM effects in patients with relapsed or refractory MM (Richardson et. al, Blood Jul 06). Significantly, a phase I clinical trial showed that lenalidomide and bortezomib could achieve responses in the majority of patients with MM, refractory to either agent alone (ASH 2005, Abstract #365). However, the effect of lenalidomide on human OCL lineage is unknown. Here we investigated the effect of lenalidomide and bortezomib on human OCL. Peripheral blood mononuclear cells (PBMC) from MM patients (n=11) and healthy donors (n=5) were stimulated with receptor activator of NFk-B ligand (RANKL) (50ng/ml) and M-CSF (25ng/ml) for two weeks to induce OCL formation, in the presence or absence of lenalidomide or bortezomib. OCL were identified by flow cytometric analysis using anti-aVb3 integrin. Lenalidomide and bortezomib inhibited OCL differentiation in a dose-dependent manner (n=13, median control: 70.9% at 0 μM; 63% at lenalidomide 2μM and 45% at 10μM; 35% at bortezomib 2nM and 11% at 5nM). TRAP staining (tartrate-resistant acid phosphatase) was performed to identify OCL and confirm OCL activity. Lenalidomide, as well as bortezomib inhibited OCL in a dose-dependent manner, as evidenced by a marked decrease in TRAP+ cells. To assess bone resorption activity, OCL were cultured with dentine discs, in the presence or absence of lenalidomide and bortezomib, followed by light microscopic analysis and additional measurement of soluble collagen I fragments from the supernatant. Both lenalidomide and bortezomib inhibited bone resorption in a dose-dependent manner. We next asked whether mature OCL were affected. OCL were induced by cytokine stimulation for 3 weeks and treated for 72h, followed by flow cytometry. Neither lenalidomide nor bortezomib altered total number of aVb3 integrin-expressing mature OCL (n=6). In addition, OCL culture supernatants were collected, and two major MM growth and survival factors produced by OCL, B-cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL), were measured by specific ELISA. Both lenalidomide and bortezomib strongly inhibited secretion of BAFF and APRIL. Finally, we determined whether lenalidomide and bortezomib inhibited expression of transcriptional factors important for OCL differentiation and survival. Cell lysates of CD14-expressing monocytic cells from normal donor PBMCs were subject to immunoblotting. Importantly, lenalidomide inhibited OCL differentiation by downregulation of PU.1 expression. These results therefore indicate, that lenalidomide and bortezomib inhibit OCL differentiation, thereby directly preventing the development of new osteolytic lesions. Moreover, BAFF and APRIL secretion by OCL is downregulated, thereby inhibiting MM cell survival in the bone marrow microenvironment.

Lenalidomide and bortezomib induce osteoclast cytotoxicity and decrease BAFF secretion in osteoclasts in human multiple myeloma: Clinical implications

7606 Background: Myeloma bone disease is caused by an enhanced osteoclast (OCL) activation, leading to osteolytic bone lesions. OCL have recently been reported to produce abundant B-cell activating factor (BAFF) in the bone marrow microenvironment that is important for myeloma cell growth and survival (Blood 106, 1021–30, 2005; ASH2005, #627). The proteasome inhibitor bortezomib (PS341, Velcade) has potent anti-myeloma activity with impressive clinical responses. A recent study indicated that bortezomib has inhibitory effects on OCL (ASH 2005, #2488). Lenalidomide (CC-5013, Revlimid) is an immunomodulatory derivative of thalidomide that has shown promising anti-myeloma effects in patients with relapsed or refractory multiple myeloma (MM) (ASH 2005, #6 &amp; #1565). However, the effect of lenalidomide on human OCL lineage is unknown. Therefore, the aim of this study is to investigate the effect of lenalidomide and bortezomib on human OCL. Methods: OCL formation from healthy donors and MM patients were stimulated with RANKL (50ng/ml) and GM-CSF (10ng/ml) in 20% FBS/RPMI. Cells were incubated with lenalidomide 2μM or bortezomib 2nM, alone and in combination, for two weeks followed by [3H]thymidine uptake assay to measure growth inhibition of OCL. Similar experiments were also performed in cocultures of CD138-purified cells from MM patients with OCL, in the presence or absence of lenalidomide and bortezomib. In addition, ELISA was performed to measure cytokine release of BAFF produced by OCL in these cultures. Results: OCL growth was decreased by lenalidomide (n=6) (median 49.2%, range 20–62.5%; coculture: 53% and 35%) as well as bortezomib (96 and 49%; coculture 63 and 59%) and by the combination of both drugs (95 and 55%; coculture 42 and 44%). BAFF secretion was dramatically decreased in cultures treated with lenalidomide 2μM (n=3) (median 72%, range 61–75.9%). Conclusions: These results indicate that lenalidomide and bortezomib inhibit OCL growth and survival. Importantly, lenalidomide and bortezomib also block growth and survival of MM cells cocultured with OCL, suggesting that lenalidomide may inhibit OCL growth and survival through inhibition of BAFF and may prevent development of osteolytic lesions in MM. No significant financial relationships to disclose.