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In multiple myeloma (MM), frequent mutations of NRAS, KRAS, or BRAF are found in up to 50% of newly diagnosed patients. The majority of the NRAS, KRAS, and BRAF mutations occur in hotspots causing constitutive activation of the corresponding proteins. Thus, targeting RAS mutation in MM will increase therapeutic efficiency and potentially overcome drug resistance. We identified germinal center kinase (GCK) as a novel therapeutic target in MM with RAS mutation. GCK knockdown (KD) in MM cells demonstrated in vitro and in vivo that silencing of GCK induces MM cell growth inhibition, associated with blocked MKK4/7-JNK phosphorylation and impaired degradation of IKZF1/3, BCL-6, and c-MYC. These effects were rescued by overexpression of a short hairpin RNA (shRNA)-resistant GCK, thereby excluding the potential off-target effects of GCK KD. In contrast, overexpression of shRNA-resistant GCK kinase-dead mutant (K45A) inhibited MM cell proliferation and failed to rescue the effects of GCK KD on MM growth inhibition, indicating that GCK kinase activity is critical for regulating MM cell proliferation and survival. Importantly, the higher sensitivity to GCK KD in RASMut cells suggests that targeting GCK is effective in MM, which harbors RAS mutations. In accordance with the effects of GCK KD, the GCK inhibitor TL4-12 dose-dependently downregulated IKZF1 and BCL-6 and led to MM cell proliferation inhibition accompanied by induction of apoptosis. Here, our data identify GCK as a novel target in RASMut MM cells, providing a rationale to treat RAS mutations in MM. Furthermore, GCK inhibitors might represent an alternative therapy to overcome immunomodulatory drug resistance in MM.

Introduction Multiple myeloma (MM)-induced bone disease remains one of its most devastating complications, caused by increased bone resorption by overactivated osteoclasts coupled with impaired bone formation. MM cells produce osteoclast-activating factors that induce osteoclast activation and extensive bone resorption. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor that is highly secreted by MM cells (Fu J etc. JCI. 2016). We also identified that the checkpoint inhibitor, programmed death-1 homolog (PD-1H/VISTA), serves as the MMP-13 receptor in osteoclasts and mediates MMP-13-dependent osteoclastogenic function which is largely blocked in Pd-1h -/-osteoclasts (Fu J etc. ASH 2019, 2020). While the inhibitory role of PD-1H/VISTA in T-cells has recently been described (ElTanbouly MA etc. Science 2020), its cellular binding proteins remain unclear, and its role in osteoclast activation andMM bone disease have not been addressed. Methods and Results To identify its interacting proteins, PD-1H-His 6 recombinant protein was expressed in mouse bone marrow mononuclear cells, the associated proteins pulled down by Ni-NTA agarose beads from cell lysates and identified by mass spectrometry. Functional annotation charting of the 75 proteins enriched in PD-1H pull-down samples (with signal ratio of PD-1H-His 6 pull-down vs control >2) indicated that almost 30% of the interacting targets were either cytoskeletal or cytoskeleton-associated proteins. Given that the F-actin cytoskeleton undergoes dynamic reorganization during osteoclast differentiation and plays critical roles in bone resorption, we further addressed the role of PD-1H in F-actin cytoskeleton regulation. Initially, osteoclasts form F-actin-rich adhesive structures, termed podosomes. At later stages, podosomes collectively rearrange into clusters and rings and finally into sealing belts to mediate osteoclast spreading, migration and bone resorption (Teitelbaum SL. Ann N Y Acad Sci. 2011). By confocal immunofluorescence microscopy, we found that PD-1H co-localized with F-actin podosome clusters, rings and sealing belts during osteoclast differentiation (Figure 1A). The functional role of PD-1H in F-actin cytoskeleton reorganization was addressed using Pd-1h -/- osteoclast wherein Pd-1h knockout lead to the disruption of podosome clusters at early stages relative to WT controls, while at later stages, Pd-1h -/-osteoclasts exhibited significantly fewer F-actin rings and belts (Figure 1B). Further, binding of MMP-13 to PD-1H increased the number of osteoclasts forming F-actin rings and belts, as well as the size of F-actin belts, which was blocked in Pd-1h -/- osteoclasts. To determine the role of PD-1H in the development of myeloma-induced lytic bone lesions, 5TGM1 myeloma cells were bilaterally intratibially injected into Pd-1h wtRag2 -/- or Pd-1h -/-Rag2 -/- mice (n=10) to induce lytic bone lesions. Three weeks following intratibial 5TGM1 injection, tibiae were harvested for micro-computed tomography. Subsequent quantitative histomorphology analyses of the trabecular and cortical bones confirmed that the knockout of Pd-1h reduced MM-induced bone destruction with significantly less decrease in trabecular bone volume (Tb. [BV/TV]), trabecular bone number (Tb. N.), trabecular bone thickness (Tb. Th.), as well as less increase in trabecular bone spacing (Tb. Sp.) and bone specific surface (Tb. [BS/BV]) compared to Pd-1h wtRag2 -/- mice. Similar effects were observed in cortical bone with less decrease in cortical bone thickness (CT. Th.), cortical bone area fraction (CT. [BA/TA]), and cortical tissue mineral density (CT. TMD) in 5TGM1 bearing Pd-1h -/-Rag2 -/- mice vs Pd-1h wtRag2 -/- mice (Table 1). Conclusions Taken together, our study, for the first time, reveals the novel role of checkpoint inhibitor, PD-1H/VISTA, in osteoclasts and myeloma bone disease. PD-1H associates with cytoskeleton proteins and regulates the F-actin cytoskeleton reorganization which is critical for osteoclast bone resorption activity. Further, PD-1H mediates MMP-13-induced osteoclast fusion, F-actin belts formation, and osteoclast activation. Pd-1h -/-in recipient mice significantly impairs MM-induced bone loss, demonstrating that PD-1H/VISTA plays a critical role in MM bone disease. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Recently, mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2) has emerged as an important key regulator of the stress-activated MAPK core signaling pathways. MAP4K2, also called Germinal Center Kinase (GCK), is predominantly and highly expressed in the germinal center of B cells. Recently we have shown that MAP4K2 knockdown in K- or N-RAS mutated MM cells induces MM cell growth inhibition, associated with the downregulation of critical transcriptional factors including IKZF1/3, BCL-6, and c-MYC proteins (Li et al. Blood 2021). Importantly, MAP4K2 silencing induces IKZF1 protein degradation without affecting IKZF1 mRNA level. Furthermore, IMiDs-resistant K-RAS Mut MM cells are sensitive to MAP4K2 inhibition induced IKZF1 degradation and cell growth suppression, suggesting that MAP4K2 inhibition overcomes IMiDs resistances in MM. To further validate MAP4K2 inhibition as a novel strategy to overcome IMiDs-resistance, we generated lenalidomide-resistant human myeloma cell lines. In this model, MM1S-LEN RES cells showed significantly decreased expression of CRBN protein compared to the parent cells. Accordingly, upon lenalidomide treatment, CRBN-mediated down-regulation of IKZF1, c-MYC, IKZF3 and IRF4 were abrogated in the MM1S-LEN RES cells. As expected, MM1S-LEN RES cells were resistant to lenalidomide induced growth inhibition in cell proliferation assay. In contrast, MAP4K2 inhibition using TL4-12 potently induced IKZF1, c-MYC, and IRF4 downregulation as well as cell proliferation inhibition, demonstrating that MAP4K2 regulates IKZF1 and cell growth independently of CRBN. These results indicate that MAP4K2 is a novel therapeutic target to overcome IMiDs-resistance MM. Iberdomide (CC-220) is an orally available IMiD® compound under development for the treatment of relapsed/refractory multiple myeloma. Previous biochemical and structural studies demonstrated that Iberdomide binds to cereblon with a higher affinity than lenalidomide or pomalidomide. Here, we evaluated the combination effects of Iberdomide with MAP4K2 silencing in MM. Tet-on sh-MAP4K2 lentivirus were introduced into RAS Mut MM cells to establish the inducible MAP4K2 knockdown cells upon doxycycline treatment. To address the combination effects, Tet-on sh-MAP4K2 RAS Mut MM cells were treated with doxycycline and different dosages of iberdomide. We found that MAP4K2 silencing strongly increased iberdomide-induced apoptosis (iberdomide alone vs. with MAP4K2 KD: 32% vs 92). Similar, in western blot assays, MAP4K2 silencing combined with Iberdomide significantly enhanced downregulation of IKZF1, c-MYC, and IRF4 compared to the iberdomide treatment alone. These data suggest that combination of iberdomide and MAP4K2 inhibition have synergetic anti-MM effects. Taken together, our findings demonstrate that MAP4K2 is a novel therapeutic target to bypass IMiDs resistance in RAS mutated MM. Combination of MAP4K2 inhibition with Iberdomide results in synergetic anti-cancer effects in MM, therefore could be a potent novel therapeutic regimen for patients with relapsed/refractory multiple myeloma. Disclosures Marcireau: Sanofi: Current Employment.

Introduction Multiple myeloma (MM) bone disease remains one of the most devastating complications of this incurable cancer, causing bone fractures, pain, mobility issues and neurological deficits. MM cells produce osteoclast-activating factors that induce osteoclast activation, thereby leading to excessive bone resorption and lytic bone lesions1. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor that is highly secreted by MM cells. MMP-13 induces osteoclast fusion and bone-resorption via a mechanism independent of its proteolytic activity2. We recently reported that MMP-13 binds to checkpoint inhibitor programmed death-1 homolog (PD-1H/VISTA), a surface receptor that is expressed in osteoclasts at high levels3. Binding of MMP-13 to PD-1H/VISTA induces osteoclast fusion and bone resorption activity whereas knockdown or knockout of PD-1H/VISTA largely block MMP-13 mediated effects on osteoclasts3. However, the function of PD-1H inMM bone disease in vitro or in vivo has not been previously defined. Methods and Results To confirm the role of PD-1H in MMP-13 induced bone disease in MM, we first conducted MM-osteoclast trans-well co-culture assay using murine MM cell line, 5TGM1 cells, and bone marrow mononuclear cells from Pd-1h-/- or wild type (WT) mice. 5TGM1 control cells or MMP-13 knockdown 5TGM1 cells were seeded in the upper wells of the transwell plates; while WT or Pd-1h-/- bone marrow mononuclear cells were seeded in the lower wells and cultured for osteoclast differentiation assessed by TRAP staining. Results show that 5TGM1 induced differentiation of WT osteoclasts with significantly increased osteoclast size and nuclei number/osteoclast. Consistent with our previous results2, MMP-13 knockdown blocked the 5TGM1 MM cells-induced activation of WT osteoclasts. In contrast, neither 5TGM1 MM cells nor MMP-13 knockdown cells had significant effects on Pd-1h-/- osteoclasts. Hence, knockout of Pd-1h abrogated MMP-13 mediated MM induction of osteoclasts, indicating that MMP-13/PD-1H interactions are critically involved in MM-induced osteoclast activation. The in vivo role of PD-1H in MM bone disease was investigated using the intratibial 5TGM1 Rag2-/- MM bone disease mice model2. For this purpose, Pd-1h-/-Rag2-/- mice were generated by crossbreeding C57BL/6 Pd-1h-/- with C57BL/6 Rag2-/- mice. 3x105 firefly luciferase expressing 5TGM1 cells (5TGM1-luc) were intratibially injected into age and sex-paired Rag2-/- or Pd-1h-/-Rag2-/- mice (N=5). Tumor progression was monitored by weekly bioluminescence imaging (BLI). 3 weeks after tumor inoculation, tibiae were harvested for quantitative micro-CT, followed by histological analysis. Histological staining showed that intratibial injection of 5TGM1-luc MM cells induced extensive lytic lesions and trabecular bone loss in Rag2-/- mice. In contrast, in Pd-1h-/-Rag2-/- mice,the bone structure was maintained with markedly less bone loss. Morphological analyses of trabecular bone across proximal tibiae further indicated that in Rag2-/- mice, 5TGM1 induced significant changes in bone microarchitecture, with decreased bone volume fraction (bone volume/tissue volume), connective density, trabecular bone numbers, and trabecular bone thickness, as well as increased trabecular bone spacing (Table 1). In contrast, in Pd-1h-/-Rag2-/- mice, 5TGM1 failed to induce significant loss of trabecular bone, confirming the critical role of PD-1H in MM induced bone disease in vivo. Conclusions Taken together, our study, for the first time, reveal that checkpoint inhibitor PD-1H/VISTA is the critical receptor for MMP-13 in osteoclasts, thereby mediating MMP-13-induced osteoclast fusion, activation and bone resorption. MM-induced trabecular bone loss was significantly lower in Pd-1h-/-mice, demonstrating that PD-1H/VISTA plays a critical role in MMP-13-induced MM bone disease. Given the checkpoint role of PD-1H/VISTA in cancer immunosuppression, we further posit that targeting the interaction of MMP-13 and PD-1H may represent a novel therapeutic strategy to treat MM bone disease and modulate the MM immune environment. References 1. Marino S, Petrusca DN, Roodman GD. Br J Pharmacol. 2019;10.1111/bph.14889. 2. Fu J, Li S, Feng R, et al. J Clin Invest. 2016;126(5):1759-1772. 3. Fu J, Li S, Yang C, et al. Blood. 2019; 134 (Supplement_1): 3072. Disclosures Lentzsch: Caelum Biosciences: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Celularity: Consultancy, Other; Magenta: Current equity holder in private company; Karyopharm: Research Funding; Mesoblast: Divested equity in a private or publicly-traded company in the past 24 months.

Introduction: RAS oncogenes are the most frequently mutated gene family in human cancers. 50% of newly diagnosed multiple myeloma patients carry a RAS/MAPK pathway mutation, with a rising percentage in the relapsed situation1. Thus, targeting RAS mutations in multiple myeloma will increase therapeutic efficiency and potentially overcome drug-resistance. Unfortunately, RAS mutations have been considered "undruggable" due to a lack of traditional small molecule binding pockets on the proteins. Therefore, key component in the RAS/MAPK pathway may represent an alternative therapeutic target for MM. Germinal center kinase (GCK), also named mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2), is an upstream activator in the MAPK pathway. Indeed, we recently discovered the critical role of GCK in RAS mutated (RASmut) MM cell survival and growth. GCK knockdown in RASmut MM cells induced MM cell growth inhibition both in vitro and in vivo. However, the detailed mechanism is yet to be defined. Methods and Results: Our previous data showed that GCK knockdown induces MM cell growth inhibition, associated with the blockage of MKK4/7-JNK phosphorylation and the downregulation of critical transcriptional factors (TFs) including IKZF1/3, BCL-6, and c-MYC proteins. To confirm that GCK knockdown downregulates IKZF1/3 etc at protein level but not mRNA level, we conducted real-time PCR on GCK knockdown MM cells and compared the expression of GCK and TFs to the empty vector (EV) infected MM cells. Results showed that shRNA induced GCK silencing only led to the significantly decreased GCK mRNA, however, did not affect IKZF1 and c-MYC expressions at mRNA level. Consistent with the effects of GCK knockdown, the GCK inhibitor TL4-12 dose-dependently downregulated IKZF1 and BCL-6 proteins, inhibited MM cell proliferation and induced cell apoptosis. IKZF1/3 are the key targets of the immunomodulatory drugs (IMiDs), which are the backbone of MM therapy. IMiDs bind to cereblon (CRBN) and induce IKZF1/3 protein degradation, which subsequently lead to MM cell growth inhibition. Importantly, our data showed that IMiDs-resistant RPMI-8226 MM cells have high expression of GCK. GCK knockdown and inhibition induced IKZF1 downregulation, triggered growth inhibition and cell apoptosis in RPMI-8226 cells, suggesting that GCK regulates IKZF1 degradation via a CRBN-independent mechanism. To confirm this hypothesis, we silenced CRBN in N-Rasmut H929 MM cells by shRNA lentiviral infection and examined the response to IMiDs and GCK inhibitor. CRBN knockdown was confirmed by western blotting. CRBN silencing in H929 cells resulted in lenalidomide (LEN) resistance, evidenced by the WTS proliferation assay. In contrast, CRBN silencing failed to rescue N-Rasmut H929 MM cells from TL4-12 induced proliferation inhibition and IKZF1 downregulation, confirming that GCK regulated IKZF1 and cell growth is independent of CRBN. Conclusion: Taken together, our data demonstrated that GCK inhibition induces cell growth inhibition and triggers apoptosis especially in RASmut MM cells. Importantly, GCK inhibitor downregulates IKZF1 via a CRBN-independent mechanism. Our findings thus provide a rationale for the clinical evaluation of targeting GCK in RASmut MM patients and further mechanistic insight into the role of GCK in MM tumorigenesis as well as drug resistance. GCK inhibitors may represent a novel therapy for the treatment of RASmut MM patients, especially those who are resistant to IMiDs as well as with refractory or relapsed MM. References Walker, B.A., et al. Mutational Spectrum, Copy Number Changes, and Outcome: Results of a Sequencing Study of Patients With Newly Diagnosed Myeloma. J Clin Oncol33, 3911-3920 (2015). Disclosures Marcireau: Sanofi: Current Employment. Lentzsch:Karyopharm: Research Funding; Mesoblast: Divested equity in a private or publicly-traded company in the past 24 months; Janssen: Consultancy; Sorrento: Consultancy; Caelum Biosciences: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celularity: Consultancy; Magenta: Current equity holder in private company; Sanofi: Research Funding.

Introduction Multiple myeloma (MM) bone disease is characterized by the development of osteolytic bone lesions due to the over-activation of osteoclast and inhibition of osteoblast cells. MM cells secret pro-osteoclastogenic factors which lead to osteoclast (OCL) activation. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor which is highly secreted by MM cells. MMP-13 induces osteoclast fusion and bone-resorption by triggering the ERK1/2-dependent up-regulation of the cell fusogen, DC-STAMP. This process operates independently of the MMP-13 proteolytic activity. The fact that MMP-13 regulates OCL signaling via a proteolytic independent mechanism suggests that MMP-13 functions as a paracrine message protein mediating the crosstalk between MM cells and OCL. However, the mechanism is yet to be identified. Methods and Results To screen for MMP-13 cellular binding proteins/receptors, we performed a MMP-13 pull-down assay wherein recombinant MMP-13-His6 was incubated with mouse mononuclear bone marrow cells (BMCs) lysates and Ni-NTA magnetic beads were used to pull-down MMP-13- associated proteins. Following mass spectral analysis, programmed death-1 homolog (PD-1H) was identified as a major MMP-13-binding protein. PD-1H, also known as V-domain Ig suppressor of T cell activation (VISTA), is a critical negative checkpoint regulator expressed on myeloid cells and involved in immune responses. Binding of MMP-13 and PD-1H was confirmed by co-expressing both proteins in HEK293 cells and subsequent co-immunoprecipitation assay. Further, following PD-1H expression in HEK293 cells, an MMP-13-GFP fusion fluorescence protein docked to the cell surface where mutagenesis studies demonstrated that the PD-1H extracellular domain (ECD) mediates the specific binding interaction. Western blot and immunohistochemistry revealed that PD-1H was highly expressed in mononuclear BMCs, pre-OCL and mature OCL. ShRNA-mediated knockdown of PD-1H in mouse mononuclear BMCs blocked the ability of MMP-13 to induce osteoclast fusion and activation. These results were further confirmed by using BMCs from Pd-1h-/- mice and WT littermates for in vitro osteoclast differentiation. While MMP-13 induced WT OCL fusion and activation, these effects were completely blocked in Pd-1h-/-OCLs in tandem with a loss in MMP-13 induced ERK1/2 phosphorylation, NFATc1 and DC-STAMP upregulation. Conclusions Taken together, our study, for the first time, revealed that checkpoint inhibitor PD-1H is highly expressed in pre- and mature osteoclast. More importantly, we identified PD-1H as the critical receptor for MMP-13 in OCL, thereby mediating MMP-13-induced OCL fusion, activation and bone resorption. Hence, MMP-13 as a novel PD-1H ligand might not only induce bone disease, but also play a potential role in the regulation of T-cell activity in MM. As such, targeting MMP-13 and PD-1H interactions may represent a novel therapeutic strategy to treat MM bone disease and modulate the MM immune environment. Disclosures Lentzsch: Abbvie: Consultancy; Clinical Care Options: Speakers Bureau; Sanofi: Consultancy, Research Funding; Multiple Myeloma Research Foundation: Honoraria; International Myeloma Foundation: Honoraria; Karyopharm: Research Funding; Columbia University: Patents & Royalties: 11-1F4mAb as anti-amyloid strategy; Bayer: Consultancy; Janssen: Consultancy; Takeda: Consultancy; BMS: Consultancy; Proclara: Consultancy; Caelum Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Introduction: Next-generation sequencing revealed frequent mutations of the RAS/mitogen-activated protein kinase (MAPK) pathway, with mutations in NRAS, KRAS or BRAF in up to 50% of newly diagnosed MM patients1. The majority of the NRAS, KRAS and BRAF mutations occur in hotspots causing constitutive activation of the corresponding pathways2. Given the upstream activator role of Germinal Center Kinase (GCK) in the MAPK pathway, GCK might be an attractive therapeutic target in MM. Indeed, we recently discovered the critical role of GCK, also named mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2), in MM cell survival and growth. Methods and Results: Our data show that GCK is higher expressed in RAS mutated MM cells compared to the wild type (WT) RAS MM cells. Silencing of GCK in RASmut MM cells (MM.1S and RPMI-8226) by using an inducible Tet-on-shGCK significantly decreased MM cell proliferation and induced cell death (Figure 1). In contrast, knockdown of GCK in RASwt MM cell lines (LP1 and U266) induced only modest inhibition of proliferation. The higher sensitivity to GCK knockdown in RASmut cells suggests that targeting GCK is especially effective in multiple myeloma which harbors RAS mutations. To exclude a potential off-target effect associated with GCK knockdown that led to the inhibition of MM proliferation, we set up an shGCK-resistant GCK allele (GCKshRNA-RES) by introducing mismatch mutations on the shRNA targeted sequences without changing the encoded amino acids. In the shGCK rescue experiment using GCKshRNA-RES we showed that all shRNA induced phenotypes (lack of growth, apoptosis and downstream effectors decrease) were corrected by the GCK resistant allele expression, ruling out the off-target hypothesis. Moreover, we expanded the in vivo studies of GCK knockdown on MM tumor progression. To monitor the tumor progression, we transduced MM.1S cells with firefly luciferase and established an inducible GCK knockdown system. Luciferase-expressing GCK inducible knockdown MM cells or non-targeting control shRNA (shCNTL) transduced MM cells were s.c. injected into SCID/Beige mice and the tumor progression was monitored by bioluminescence imaging. Doxycycline (for induction of shRNA) or vehicle treatment were started after the tumor was established on day 16 to induce shGCK and subsequently silence GCK expression. In contrast to the vehicle-treated MM.1S-Tet-on-shGCK or doxycycline-treated MM.1S-Tet-on-shCNTL tumors, doxycycline-treated animals bearing MM.1S-Tet-on-shGCK xenografts showed a significant inhibition (P Lysine 45 is critical for GCK kinase activity. Point mutation of K45A will completely abolish its kinase activity. We introduced K45A mutation into GCKshRNA-RES (GCKshRNA-RESK45A→ shGCK resistant and kinase dead GCK). Tet-on-shGCK with GCKshRNA-RES or GCKshRNA-RESK45A were co-transduced in MM.1S cells. As expected, the GCK knockdown effects were rescued by GCKshRNA-RES but not by the kinase-dead mutant GCKshRNA-RESK45A. In contrast to GCKshRNA-RES, GCKshRNA-RESK45A failed to stimulate MM cell proliferation, to suppress MM cells apoptosis and to restore the downstream effectors expression. Our findings demonstrated that GCK kinase activity is required for its function in myeloma cell physiology. Conclusion: Taken together, our findings provide a rationale for the clinical evaluation of targeting GCK in MM patients and the role of GCK in MM tumorigenesis as well as drug resistance. The subsequent development of small molecules inhibiting this pathway, such as GCK kinase inhibitors, will address the unmet need of developing targeted treatments for RASmut myeloma and potentially for other RASmut malignancies. References 1. Walker, B.A., et al. Mutational Spectrum, Copy Number Changes, and Outcome: Results of a Sequencing Study of Patients With Newly Diagnosed Myeloma. J Clin Oncol33, 3911-3920 (2015). 2. Xu, J., et al. Molecular signaling in multiple myeloma: association of RAS/RAF mutations and MEK/ERK pathway activation. Oncogenesis6, e337 (2017). Disclosures Marcireau: Sanofi: Employment. Lentzsch:Caelum Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Takeda: Consultancy; BMS: Consultancy; Proclara: Consultancy; Abbvie: Consultancy; Clinical Care Options: Speakers Bureau; Sanofi: Consultancy, Research Funding; Multiple Myeloma Research Foundation: Honoraria; International Myeloma Foundation: Honoraria; Karyopharm: Research Funding; Columbia University: Patents & Royalties: 11-1F4mAb as anti-amyloid strategy; Bayer: Consultancy.

Introduction: c-Myc is a master transcription factor and one of the most frequently altered genes across a vast array of human cancers including diffuse large B-cell lymphoma (DLBCL), and is thus an attractive therapeutic target . However, no direct inhibitor of c-Myc has been successfully developed for the treatment of any cancer. The c-Myc protein has a short half-life of less than 30 minutes , and the complex secondary structures in the 5' untranslated region (UTR) of MYC mRNA make its translation highly dependent on the eukaryotic translation initiation factor 4F (eIF4F) . eIF4F exists as a complex comprised of the eIF4E, eIF4A, and eIF4G subunits. eIF4E can be sequestered by 4E-BP1, which acts as a "brake" for initiation of mRNA translation . Hyper-phosphorylation of 4E-BP1, caused by upstream signals such as mTORC1, leads to release of eIF4E from 4E-BP1, assembly of the eIF4F complex, and robust mRNA translation. Surprisingly, neither FDA approved mTORC1 inhibitors nor the investigational mTORC1/mTORC2 inhibitor MLN0128 has demonstrated adequate activity in aggressive lymphoma. The therapeutic effects of mTOR inhibition in c-Myc driven aggressive lymphoma remain poorly understood. Recognizing phosphoinositide 3-kinase (PI3K) and the proteasome pathway are both involved in activating mTOR, we hypothesized that co-targeting the PI3K and proteasome pathways might synergistically inhibit translation of c-Myc. Since both PI3K and proteasome are proven drug targets in blood cancer, such co-targeting strategy may be expeditiously studied in clinical trials for c-Myc driven aggressive lymphoma. Methods: Cytotoxicity was studied in lymphoma cell lines and primary lymphoma cells using Cell TiterGlo (Promega®). The Bliss additivism model was used to determine the expected inhibition of cell growth and the excess over Bliss (EOB) values. EOB values above 0 indicate synergy, with higher values indicating higher levels of synergy. Expression of c-Myc was investigated at the translation and transcription levels, using a combination of Western blot, qPCR, and a bi-cistronic luciferase reporter we developed to study cap dependent translation. Gene expression profiling (GEP) studies were conducted using RNAseq, and analyzed by the Fisher t-test and running enrichment score (RES) between different treatment groups. Mechanisms of synergy were determined through interrogating the effects of small molecule inhibitors and shRNA targeting regulators of various regulators of 4E-BP1. Structural studies of TGR-1202 were performed by in silico docking, and validated by synthesis of novel analogs of TGR-1202. Activity of TGR-1202 on CK1 epsilon was studied by kinome profiling (Reaction Biology®), cell free kinase assay of CK1e (Promega®), and cell based assay of CK1e autophosphorylation. Results: We found that a novel PI3K delta isoform inhibitor TGR-1202, but not the approved PI3Kd inhibitor idelalisib, was highly synergistic with the proteasome inhibitor carfilzomib in lymphoma, leukemia, and myeloma cell lines and primary lymphoma and leukemia cells (Figure 1). TGR-1202 and carfilzomib (TC) synergistically inhibited phosphorylation of eIF4E-binding protein 1 (4E-BP1), leading to suppression of c-Myc translation and silencing of c-Myc dependent transcription (Figure 2). Furthermore, the synergistic cytotoxicity of TC was rescued by overexpression of eIF4E or c-Myc. TGR-1202, but not other PI3Kd inhibitors, was active against casein kinase-1 (CK1) epsilon (Figure 3). Targeting CK1e using a selective chemical inhibitor or shRNA complements the effects of idelalisib, as a single agent or in combination with carfilzomib, in repressing phosphorylation of 4E-BP1 and the protein level of c-Myc. Conclusion: These results suggest that TGR-1202 is a first-in-class dual PI3Kd/CK1e inhibitor, which may in part explain the preliminary clinical activity of TGR-1202 in aggressive lymphoma not found with idelalisib. Targeting CK1e should become an integral part of therapeutic strategies targeting translation of oncogenes such as c-Myc. Disclosures Lentzsch: BMS: Consultancy; Foundation One: Consultancy; Celgene: Consultancy, Honoraria. O'Connor:Seattle Genetics: Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Research Funding; Spectrum: Research Funding; Spectrum: Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; TG Therapeutics: Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Celgene: Research Funding; Celgene: Research Funding.

IFNγ signaling plays a critical role in the pathogenesis of GVHD. In this study, we observed that LPS-maturated bone marrow-derived dendritic cells (BMDCs) lacking IFNγ receptor (IFNγR, GRKO) or signal transducer and activator of transcription 1 (STAT1KO) had increased expression of major histocompatibility complex class II (MHC II), CD86, CD80, and enhanced allo-stimulatory capacity. This was further confirmed using fully MHC-mismatched bone marrow transplantation (BMT) studies. APC of GRKO or STAT1KO recipients had increased MHC II expression, which was associated with enhanced activation and expansion of donor CD4 and CD8 T cells and subsequently accelerated GVHD mortality compared to wild type (WT) controls. This increased GVHD mortality and increased MHC II expression on host APCs was further observed in the absence of recipient conditioning in the B6→CB6F1 mouse model. This was associated with increased presentation of host derived endogenous Eα52-68 peptide via I-Ab on recipient CD11c+ cells as detected by staining with the YA-e antibody. Furthermore, we could demonstrate that absence of IFNγR in BMDC promotes presentation Eα52-68 peptide and subsequently elicits pronounced activation, expansion and Th1 differentiation of TEa-TCR-tg CD4 T cells which recognize the Eα52-68 peptide presented by I-Ab. Next, we assessed the impact of this pathway on presentation of exogenous antigens. Interestingly, when lysate prepared from BALB/c splenocytes was incubated with BMDCs from B6 mice, Y-Ae expression on STAT1-/- BMDCs was significantly reduced compared to wild type BMDCs allowing us to hypothesize that IFNγ/STAT1 signaling may play an important role in promoting presentation of exogenous antigen while suppressing presentation of endogenous antigen. To further confirm this hypothesis, we used ovalbumin (OVA) as a second model antigen. To assess the impact of IFNγ/STAT1 signaling on presentation of exogenous antigen, WT, GRKO or STAT1KO BMDC were directly pulsed with OVA. To address the role in endogenous antigen presentation we studied act-mOVA-transgenic wildtype, act-mOVA.GRKO or act-mOVA.STAT1KO BMDCs. Transgenic OT-II CD4 T cells express a TCR specific for the OVA peptide 323-33 presented by I-Ab. The proliferation/activation of OT II T cells was monitored by flow cytometer as readout for effective Ag presentation. Our data demonstrated that IFNγR- or STAT1-deficient BMDCs loaded with exogenous intact OVA protein were compromised in promoting OT II proliferation. In contrast, responder OT-II CD4 T cells proliferated much more vigorously when stimulated with IFNγR/STAT1-deficient m-Act-OVA BMDCs compared to controls. We further observed significantly impaired OT-II cell proliferation in IFNγR or STAT1-deficient mice immunized with OVA indicating impaired presentation of exogenous antigens. However, OT-II CD4 T cells injected into lethally irradiated act-mOVA.STAT1KO transgenic mice proliferated more robustly and displayed increased Th1 differentiation compared to control mice when tested 3 days after OT II administration. We next started to assess several key factors (Ii [invariant chain, CD74], Cathepsin S [CTSS], H2-M, CIITA and MARCH1), known to be involved in the process of MHC class II antigen presentation and MHC II expression. We found retention of Invariant chain (CD74) expression as well as reduced CTSS and H2M expression in GRKO or STAT1KO BMDC following LPS-maturation. Furthermore, we observed significantly reduced lysosome formation/function in STAT1KO BMDCs compared to wild type BMDCs after LPS maturation. These data suggest that exogenous protein-derived peptide exchange in the MHCII compartment (MIIC) is impaired in STAT1KO BMDCs. Immature and LPS-maturated STAT1-/-BMDCs had significantly increased autophagy, which could explain enhanced endogenous Ag presentation since autophagy has been demonstrated to be critical in MHC II Ag presentation of cytoplasmic constituents. Finally, we found evidence of enhanced MHC II synthesis as supported by increased CIITA mRNA expression and conversely reduced MHC II degradation as indicated by reduced MARCH1 expression. In summary our data suggest that absence of IFNγR/STAT1 signaling in DC leads to abnormal surface MHC II turnover, promotes presentation of endogenous peptides and concomitantly impairs processing and presentation of exogenous antigens. Disclosures Lentzsch: BMS: Consultancy; Foundation One: Consultancy; Celgene: Consultancy, Honoraria.

Introduction: Bay 61-3606 is a cell-permeable imidazopyrimidine compound that acts as a potent, ATP-competitive, reversible, and highly selective inhibitor of Syk tyrosine kinase activity with no inhibitory effect against Btk, Fyn, Itk, Lyn, and Src. BAY 61-3606 has been also shown to inhibit Syk-mediated cellular functions such as glucose-tyrosine phosphorylation of I κ B α and p65 nuclear translocation. It further exhibits a good oral bioavailability and in vivo efficacy in rat models. Recently, Bay 61-3606 was found to inhibit cell proliferation and SDF-1a-induced migration of MM cells (1). Based on these promising preliminary data we further investigated the potential of Bay 61-3606 as new anti-MM agent. Methods and Results: Bay 61-3606, at concentrations as low as 10 nM, induced significant (p Conclusion: In summary, our results demonstrate that Bay 61-3606 has anti-MM effects by inducing cell cycle arrest and apoptosis, and this effect is independent of SYK inhibition. Similar to IMiD® compounds, Bay 61-3606 induces IKZF1 and IKZF3 protein degradation in MM cells. More importantly, Bay 61-3606 has effects on both IMiD®-sensitive and resistant myeloma cells and may represent a novel potent anti-MM agent. References: (1). Koerber RM, et al. Exp Hematol Oncol. 2015. Disclosures Lentzsch: BMS: Consultancy; Foundation One: Consultancy; Celgene: Consultancy, Honoraria.

Background : We have recently shown that Matrix metalloproteinase-13 (MMP-13) is highly expressed in human multiple myeloma (MM) cells and MMP-13 increases osteoclast (OCL) fusion and bone resorption independently of its metalloproteinase activity. In a mouse model of MM bone disease, knockdown (KD) of MMP-13 in MM cells inhibited development of bone lytic lesions, suggesting that MMP-13 is a potential therapeutic target for the treatment of MM bone disease (MMBD). However, the effects of MMP-13 on the MM bone marrow (BM) niche and underlying mechanism by which MMP-13 induces OCL fusion remains undefined. Methods and Results: To delineate the effects of MMP-13 on MM BM microenvironment, we investigated the effects of MMP-13 on cell proliferation and differentiation of pre-OCL, pre-osteoblast and MM cells. WT or Mmp-13-/- mouse CD11b+ BM cells were cultured with or without MMP-13 for 3 days and pre-OCL proliferation was evaluated by WST-1 assay. Neither MMP-13 treatment nor Mmp-13 deficiency affected pre-OCL proliferation, indicating MMP-13 induces OCL activation rather by promoting cell fusion than by affecting cell proliferation. To test the effects of MMP-13 on osteoblast (OB) differentiation and mineralization, mouse pre-OB cell line MC3T3-E1 and primary pre-OB cells from WT or Mmp-13-/- mice BM were cultured with or without MMP-13 for 7-14d. Neither exogenous MMP-13 nor Mmp-13 knockout affected OB differentiation or mineralization in vitro. Consistent with the in vitro observation, our Rag2-/- mouse 5TGM1 MMBD model provided in vivo evidence that knockdown of MMP-13 in MM cells does not affect MM induced OB inhibition or impairment of bone formation. Further, the effects of MMP-13 on MM cell proliferation were also assessed both in vitro and in vivo. Treatment of human MM cell lines RPMI-8266, OPM2 and MM.1S, as well as mouse MM cell line 5TGM1 with MMP-13 for up to 3 days did not induce in vitro cell proliferation. Similarly, MM cell sensitivity against anti-MM agents bortezomib and pomalidomide was not affected by MMP-13 in vitro. But, MMP-13 KD in mouse 5TGM1 MM cell line caused significantly less in vivo tumor burden in Rag2-/- intratibial MMBD model compared to 5TGM1 empty vector control cells, which we speculate occurs as a consequence of effects secondary to decreased OCL activation within the bone microenvironment following MMP-13 silencing. Our previous experiments showed that MMP-13 induces OCL fusion independent of its enzymatic activity, however the underlying mechanism is unknown. To further analyze the mechanism of action immunofluorescence staining and cell fraction-immunoblotting assay demonstrated that extracellular MMP-13 undergoes cell surface binding and subsequent active intracellular translocation into the perinuclear area, further supporting the enzymatic activity independent mechanism of MMP-13 on OCL induction. Furthermore, analysis of MMP-13-induced signaling pathways mediating osteoclastogenesis revealed that MMP-13 activates multiple cell signaling pathways including MAPK, PI3K and NF-kappaB independent of its enzymatic activity. Interestingly, the noncanonical NF-kappaB pathway is selectively activated by either MMP-13 WT or an E223A catalytically-inactive mutant, but not any by any other MMPs or MMP-13 truncated non-functional mutants, indicating that signaling through the noncanonical NF-kappaB pathway in OCL is specific for MMP-13 and may play a role in MMP-13-driven OCL fusion. Further studies are underway to screen for MMP-13 receptors and binding partners in an effort to delineate the processes underlying MMP-13 signaling. Conclusion: Our results demonstrate that MM produced MMP-13 induces OCL activation and bone resorption without affecting OB inhibition and bone formation impairment as well as anti-neoplastic response of MM cells. MMP-13 silencing in MM protects bone structure, and inhibits MM progression in vivo, which may be secondary to decreased OCL activation. MMP-13 specifically acts on OCL fusion. Signaling study showed noncanonical NF-kappaB signaling is selectively activated by MMP-13 independently of its enzymatic activity, and may be critical for MMP-13-induced OCL fusion and activation. Further screening the potential cellular receptor and intracellular transportation mechanism of MMP-13 in OCL is ongoing. Disclosures Roodman: Amgen: Consultancy; Eli Lilly: Research Funding. Lentzsch:Axiom: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

Background: c-Myc is one of the most frequently altered genes across a vast array of human cancers. Overexpression of c-Myc is observed in up to 30% of cases of diffuse large B-cell lymphoma (DLBCL), the most common type of aggressive lymphoma. Although DLBCL can be cured in 60-70% patients, a substantial minority of patients with DLBCL still die from their lymphoma. There is emerging evidence that c-Myc expression is an adverse risk factor independent of the cell-of-origin classification. To date no drugs that directly target c-Myc have been approved for the treatment of any cancer. In fact, since c-Myc is involved in many normal cellular functions, direct c-Myc inhibitors may be associated with significant toxicity. The goal of our study is to develop novel strategies targeting c-Myc that will have an improved therapeutic index. The c-Myc protein has a very short half-life of less than 30 minutes, and its translation is highly dependent on the eukaryotic initiation factor 4F (eIF4F). eIF4F is activated by the mammalian target of rapamycin (mTOR), which is regulated not only by the PI3K-AKT pathway but also the proteasome pathway. These observations led us to hypothesize that if the proteasome and PI3K pathways cooperate in the activation of mTOR and its downstream target eIF4F, then combinations of proteasome and PI3K inhibitors should potently suppress eIF4F dependent translation of c-Myc and the growth of c-Myc dependent lymphoma. Methods: Cytotoxicity was studied in lymphoma cell lines and primary lymphoma cells using Cell TiterGlo. The Bliss additivism model was used to determine the expected inhibition of cell growth. The difference of the expected and observed levels of inhibition was used to calculate the excess over Bliss (EOB) values. EOB values above 0 indicate synergy, with higher values indicating higher levels of synergy. Mechanisms of synergy were determined through interrogation of PI3K/AKT/mTOR pathway and its downstream targets. Expression of c-Myc was investigated at the translation and transcription levels, using a combination of Western blot, qPCR, and a bi-cistronic luciferase reporter we developed to study cap dependent translation. Gene expression profiling (GEP) studies were conducted using RNAseq, and analyzed by the Fisher t-test and running enrichment score (RES) between different treatment groups. Results: We used a high-throughput platform to screen the cytotoxicity of two PI3Kdelta inhibitors (TGR-1202 & idelalisib/Cal-101), two proteasome inhibitors (bortezomib & carfilzomib), and four combination pairs using these drugs. We found that TGR-1202 and Cal-101 caused only minimal to mild inhibition of lymphoma cells, while bortezomib and carfilzomib caused potent inhibition as single agents. The combination of TGR-1202 and carfilzomib was consistently the most synergistic doublet, while the combination of Cal-101 and bortezomib the least synergistic in numerous lymphoma cell lines studied to date (Figure 1). TGR-1202 and carfilzomib were also highly synergistic in primary lymphoma cells, while Cal-101 and bortezomib were not. Importantly, normal lymphocytes were resistant to the combination of TGR-1202 and carfilzomib. At the molecular level, only the combination of TGR-1202 and carfilzomib potently inhibited mTORC1 dependent phosphorylation of 4E-BP1, leading to marked reduction of c-Myc protein (Figures 2 & 3). In contrast, the combination of TGR-1202 and carfilzomib produced no reduction of the mRNA level of c-Myc (Figures 3). A luciferase reporter demonstrated that the synergistic combination TGR-1202 and carfilzomib specifically inhibited the translation downstream of the 5'UTR of c-Myc (Figure 3). GEP studies confirmed that the canonical c-Myc target genes were potently downregulated at the level of transcription by the combination of TGR-1202 and carfilzomib (Figure 3). These results demonstrate that TGR-1202 and carfilzomib in combination potently inhibited the translation of c-Myc and the c-Myc transcriptional program, which appears to be primarily through disruption of the PI3Kdelta and proteasome pathways that converge on mTOR. Ongoing experiments are focused on confirmation of these observations in vivo. Further, a phase I/II clinical trial evaluating this combination regimen in aggressive c-Myc driven lymphomas is being planned. Disclosures Deng: TG Therapeutics: Research Funding; Gilead: Research Funding; Amgen/Onyx: Research Funding. Lentzsch:Celgene: Consultancy; BMS: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Axiom: Honoraria. O'Connor:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Research Funding; Acetylon: Consultancy, Other: Consultancy fee; Spectrum Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb Company: Consultancy, Other: Consultancy fee; Takeda Millenium: Consultancy, Honoraria, Other: Consultancy fee, Research Funding; Novartis: Consultancy, Honoraria, Other: Consultancy fee; Seattle Genetics: Research Funding; Bayer: Consultancy, Honoraria.

Introduction: Previously we have shown that the immune modulatory drugs (IMiDs) downregulate GATA1 and PU.1 resulting in maturational arrest of granulocytes with accumulation of immature myeloid precursors and subsequent neutropenia. Our studies further revealed that similar to MM cells cereblon (CRBN) is critical for the mediation of the effects of IMiDS in hematopoietic stem cells (HSCs) and associated with decrease of IKZF1-dependent transcription factors such as GATA1 and PU.1, which are critical for development and maturation of neutrophils and erythrocytes as well as thrombocytes. Here we investigated the mechanism how IMIDs induce degradation of IKZF1 and confirmed our studies in vivo by using the humanized NOD/SCID/Gamma-c KO (NSG) mouse model. Methods and Results After we had shown that knockdown of CRBN in HCS mediates resistance to IMIDs (2014 ASH abstract 418) we assessed the impact of IKZF1 inhibition using two different approaches. First, we knocked down IKZF1 expression in CD34+ cells by shRNA lentivirus transduction. As expected, IKZF1 knockdown in CD34+ cells mimicked the effects of IMiDs resulting in increased CD34+ cell proliferation, CD33+ cell expansion (flow cytometry) and shift of lineage commitment from BFU-E to CFU-G (colony assay). Knockdown of IKZF1 was associated with decreased GATA1 and PU.1 expression at both mRNA and protein levels. Next, we generated a mutant IKZF1 by substituting Glutamine Q146 to Histidine, which abrogates IKZF1 ubiquitination induced by CRBN. CD34+ cells were transduced with lentiviral constructs to overexpress IKZF1-WT or IKZF1-Q146H. POM failed to induce IKZF1 degradation in IKZF1-Q146H-OE CD34+ cells, indicating CRBN binding to IKZF1 and subsequent ubiquitination is critical in this process. Functional assays further confirmed that IKZF1-Q146H CD34+ cells were resistant to POM induced CD33+ cell expansion and shift in lineage commitment from BFU-E to CFU-G. Since conventional mouse models are not applicable to test IMIDs in vivo due to the fact that IMIDs do not bind to mouse CRBN (Kronke, Fink et al. 2015), we established a humanized mouse model resembling human hematopoiesis. In this model, NOD/SCID/Gamma-c KO (NSG) mice received human fetal thymus grafts and 105 CD34+ fetal liver cells to generate human hematopoiesis including functional T-cells. After establishing human hematopoiesis mice were injected with POM (0.3 mg/kg) i.v every 2 days for 3 weeks. Analysis of bone marrow revealed that POM treatment significantly induced granulocyte/macrophage progenitor cells (CD34+ CD38+ CD45RA+ cells) at the expense of common lymphoid progenitors (CD34+ CD10+ cells). The shift into myelopoiesis is consistent with our in vitro finding that IMiDs affect lineage commitment. Conclusion: In summary, our results demonstrate that IMiDs affect CD34+ cell fate via CRBN and IKZF1 mediated mechanism. These results will be helpful to elucidate the mechanism of IMiDs on lineage commitment and maturation in HSCs. Also establishment of the humanized xenograft mice model may provide an advanced platform for the analysis of human hematopoiesis and human immune responses to IMiDs as well development of secondary hematologic malignancies in vivo. Disclosures Lentzsch: Axiom: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

The mechanisms by which the human T-cell leukemia virus type I (HTLV-I) Tax oncoprotein deregulates cellular signaling for oncogenesis have been extensively studied, but how Tax itself is regulated remains largely unknown. Here we report that Tax was negatively regulated by PDLIM2, which promoted Tax K48-linked polyubiquitination. In addition, PDLIM2 recruited Tax from its functional sites into the nuclear matrix where the polyubiquitinated Tax was degraded by the proteasome. Consistently, PDLIM2 suppressed Tax-mediated signaling activation, cell transformation, and oncogenesis both in vitro and in animal. Notably, PDLIM2 expression was down-regulated in HTLV-I–transformed T cells, and PDLIM2 reconstitution reversed the tumorigenicity of the malignant cells. These studies indicate that the counterbalance between HTLV-I/Tax and PDLIM2 may determine the outcome of HTLV-I infection. These studies also suggest a potential therapeutic strategy for cancers and other diseases associated with HTLV-I infection and/or PDLIM2 deregulation.

Introduction: Lenalidomide (LEN) and pomalidomide (POM) are derivatives of thalidomide (IMiD® compounds) and currently used to treat multiple myeloma (MM) and B-cell malignancies. We have shown before that IMiD® compounds shift lineage commitment of CD34+ cells towards myeloid development by affecting critical transcription factors such as GATA1 and PU.1 with concomitant inhibition of cell maturation resulting in anemia and neutropenia. Nonetheless the underlying pathomechanism is still unknown. Recently, IMiD® compounds were shown to bind to cereblon (CRBN) in MM cells, which is the substrate recognition component of cullin-dependent ubiquitin ligase and LEN leads to ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IZKF3 by the CRBN-CRL4 ubiquitin ligase. We investigated here the role of CRBN, IKZF1 and IKZF3 in IMiD®-induced effects on lineage commitment and maturation of CD34+ cells. Methods and Results: By western blot analysis we found that CRBN and IKZF1, but not IKZF3 are expressed in CD34+ cells. Treatment of CD34+ cells with LEN and POM for only 1h almost completely decreased the expression of IKZF1 without affecting CRBN protein expression. By using a thalidomide analog bead assay, we found that IMiD® compounds directly bind CRBN in CD34+ cells. In contrast to our protein studies, IKZF1-mRNA level was not altered in real-time PCR, suggesting that IMiD® compounds regulate IKZF1 at post-transcriptional level. Treatment with proteasome inhibitors MG132, PS341 or MLN4924 which function as cullin-dependent ubiquitin ligase inhibitors blocked LEN and POM induced IKZF1 degradation, confirming that IMiD® compounds induce ubiquitination and subsequent protein degradation of endogenous IKZF1. Next we generated lentiviral constructs to knockdown the expression of CRBN in CD34+ cells. Knockdown of CRBN in CD34+ cells induced resistance to POM-induced IKZF1 downregulation and subsequently reversed the POM-induced lineage shift in colony-formation assays, suggesting that POM-induced degradation of IKZF1 in HSC requires CRBN.Knowing that PU.1 and GATA-1 are critically involved in the IMID-induced lineage shift in CD34+ cells we demonstrated in chromatin immunoprecipitation assays that IKZF1 binds to promoter regions of PU.1 and GATA-1, suggesting that PU.1 and GATA-1 are direct downstream targets of IKZF1 in CD34+ cells . Conclusion: Our findings show that CRBN and IKZF1 mediate the effects of IMiD® compounds on hematopoietic progenitors. IMiD® compounds promote CRBN dependent degradation of IKZF1 protein in CD34+ cells that subsequently decreases transcription factors such as GATA1 and PU.1 which are critical for development and maturation of neutrophils and erythrocytes as well as thrombocytes. Our findings that IMiD® compounds mediate their effects via CRBN and IKZF1 provide for the first time the pathomechanism how LEN and POM affect hematopoiesis and induce neutropenia, thrombocytopenia as well as anemia. Disclosures Lentzsch: Bristol Myers Squibb: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Research Funding.

Introduction:IMiD® compounds such as lenalidomide (LEN) and pomalidomide (POM) represent a novel class of agents in the treatment of multiple myeloma. Increasing concerns about its potential toxicity on stem cells resulting in an increase of secondary malignancies primarily affecting the myeloid cells have been reported. However, the exact mechanism is unclear. MicroRNAs (miRNAs) are emerging as important regulators in stem cell development. Therefore, the present study investigated the effect of IMiD® compounds on stem cell renewal/proliferation and the role of miRNAs involved in this process. Methods and Results: When human CD34+ cells were cultured with POM or DMSO under conditions favoring myeloid development, POM significantly increased the percentage of CD34+cells in S phase in cell cycle analysis with concomitant dramatic decrease of apoptotic cells. This resulted in a strong and significant proliferation of CD34+cells as well as induction of colony formation. Systematic screening of miRNAs expression profile by microarray showed that POM significantly enhanced the expression of miR-125b. Upregulation of miR-125b was further confirmed by realtime PCR. Interestingly, recently miR-125b was shown to function as an oncomir by inducing tumorigenesis in myeloid cells by So et al., Blood 2014. In accordance with that we found that overexpression of miR-125b in CD34+ cells exhibited similar effects as POM on CD34+ cells, such as increased cell number in S-phase, decreased cell apoptosis resulting in significant induction of colony formation and the absolute number of hematopoietic progenitors. Bioinformatics prediction models revealed a potential binding site of miR-125b on 3'UTR of P53. Indeed subsequent experiments showed that P53 was downregulated when miR-125b was overexpressed in CD34+cells or when treated with POM. Further analysis of the P53 down stream targets revealed that POM decreased the expression of PUMA and BAK1 all critical for control of cell proliferation and apoptosis. The POM-induced upregulation of mir-125b was independent of the CRBN/IKZF1 induced protein degradation. Conclusion: Based on our results we postulate that POM upregulates the oncomir miR-125b in CD34+cells. Mir-125b controls proliferation and apoptosis by downregulating P53 and genes involved in the P53 pathway including BAK1 and PUMA. Thus, our findings on the effects of POM on the miR-125b/P53 axle provide for the first time a potential mechanism for the development of secondary hematologic malignancies in patients treated with IMiD® compounds. Disclosures Lentzsch: Celgene: Consultancy, Research Funding; Novartis: Consultancy; Bristol Myers Squibb: Consultancy.

Interferon-gamma (IFN-γ) is a pleiotropic cytokine that has diverse activities in mediating host defense and immunopathology and is involved in both innate an adaptive immunity. Furthermore, IFN-γ has been shown to be a key regulator of GVHD. STAT1 is the major downstream signal transducer for IFN-γ. IFN-γ has been shown to up-regulate MHC class II expression on macrophages and dendritic cells. In this study, we unexpectedly observed that STAT1-deficient mouse bone marrow-derived dendritic cells (BM-DCs) displayed significantly increased MHC class II, CD86, CD80, CD40 expression compared to wild type BM-DCs upon LPS stimulation. To determine whether these LPS-maturated STAT1-/- BM-DCs are functional, freshly isolated pan-T cells from BALB/c (H2d) mice were stimulated with either irradiated LPS-maturated STAT1+/+ or STAT1-/- BMDCs from 129Sv [H2b] mice at different DC/Responder ratios for 5 days. Pan-T cell proliferation was measured using by 3H-incorporation or in separate assays by CFSE dilution. Both proliferation assays clearly demonstrated that STAT1-/- BM-DCs were significantly more potent stimulators in Mixed Lymphocyte Reaction (MLR) assays compared to STAT1+/+ BM-DCs. Further studies demonstrated that this increased surface MHC II expression was also observed in LPS-stimulated IFNγR-/- BM-DCs or on wild type BM-DCs treated with neutralizing IFNγ Abs but not in LPS-treated IFNα-R-/- BM-DCs. STAT1 deficient DCs have been reported to be defective in IL-12 production upon stimulation with various Toll-like receptors (TLRs). However, we were able to observe that STAT1-/- BM-DCs had increased CD40 expression after LPS maturation, and more importantly, STAT1 deficient BM-DCs had significantly increased IL-12 production upon treatment with CD40-ligand. These data suggest that STAT1 deficiency in LPS-stimulated DCs results not only in increased expression of MHC class II and costimulatory molecules, but also in enhanced CD40-dependent IL-12 production promoting generation of Th1 and Tc1 cell of wildtype responder cells. To test the influence of IFNγ/STAT1 deficiency in host APC on GVHD induction, wild type or STAT1-deficient 129 mice (H2b) underwent allogeneic Bone Marrow Transplantation (BMT) following lethal irradiation (1044 rad) receiving BALB/c spleen cells. 129.STAT1-/- recipients had significantly accelerated GVHD mortality (MST 8 days vs. 5 days, log rank test p=0.004) compared with wild type recipients. In contrast, STAT1-deficient syngeneic controls did not show enhanced mortality or morbidity ruling out that the observed accelerated mortality following allogeneic BMT was due to conditioning toxicity. On day+4 post-BMT animals were sacrificed and splenocytes were analyzed by flow cytometry. We could observe a significantly enhanced expansion and activation of donor CD4 and CD8 T cells in STAT1-/- recipients. Importantly, the increased MHC II expression was confirmed in host CD11b+ and CD11c+ cell populations. To clarify that STAT1 deficiency in host APCs do contribute to increased GVHD induction, we generated STAT1-/-→STAT1+/+, and STAT1+/+→STAT1+/+ radiation chimeric mice as recipients so that only the hematopoietic cells were STAT1 deficient. We again observed significantly increased GVHD mortality and increased activation of fully MHC-mismatched donor T cells in STAT1-/-→STAT1+/+ recipients following allogeneic BMT and GVHD induction. Similar results i.e. increased GVHD mortality, increased donor cell activation and increased MHC II expression on host CD11c+, B220+ cells were also found in IFNγR-/- recipient mice when compared with control mice following full MHC-mismatched allogeneic BMT. In summary our data reveal previously unknown effects of IFNγ/STAT1 signaling on APCs function suggesting that LPS-driven maturation of APC’s is negatively regulated by IFNγ/STAT1 leading to enhanced expression of MHC class II, costimulatory molecules and responsiveness to CD40-ligation and subsequent IL-12 production in the absence of STAT1. Furthermore, our results explain the accelerated GVHD induction and demonstrate that IFN-γ/STAT1 signaling in host APC mitigates the development of GVHD. Disclosures: Lentzsch: Celgene: Research Funding.

Introduction Overexpression and/or activation of eukaryotic initiation factor 4E (eIF4E) is critical for oncogenic protein synthesis. Mutations in genes related to mRNA translation are involved in the pathogenesis of multiple myeloma (Chapman, Lawrence et al. 2011). Recently, we found that MM cells express high levels of eIF4E protein compared to normal plasma cells and overexpression of eIF4E induces transcription factors such as c-myc critical for the growth of multiple myeloma cells (Li, Fu et al. 2011,2012). The understanding of the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. Here we show the critical role of eIF4E driven protein synthesis by using an inducible knockdown system to silence eIF4E gene expression and confirm the critical role of eIF4E in multiple myeloma growth in vivo and in vitro. Methods and Results We stably infected U266, RPMI-8226, IM-9 and MM.1S cells with a robust inducible single-lentiviral knockdown vector pLKO-Tet-On containing either control non-targeting shRNA or eIF4E targeting shRNA sequences. Doxycycline-induced eIF4E shRNA expression resulted in significant decrease of eIF4E mRNA and protein in eIF4E-shRNA but not the control shRNA infected MM cells. To determine the effects of eIF4E knockdown on MM cell growth and viability, stably transfected cell lines were grown in the presence or absence of doxycycline. Silencing of eIF4E by doxycycline induction of eIF4E shRNA in RPMI-8226 cells significantly inhibited (>72%,P Conclusion Here we show that eIF4E, a key player in the translational machinery, promotes multiple myeloma cell growth. We found that high eIF4E expression is indispensable for the growth of MM cells both in vitro and in vivo. Silencing of eIF4E decreases protein expression of a subset of transcripts encoding regulators of the cell cycle and proliferation, and resulted in tumor inhibition. Our study indicated that targeting transcriptional initiating factor eIF4E may represent a novel therapeutic strategy for MM treatment. Disclosures: Schecter: Seattle Genetics: Honoraria, Research Funding. Lentzsch:Celgene: Research Funding.

Abstract 4025 Background: MM cells produce several osteoclast-activating factors, which result in highly activated osteoclasts and cause dysregulated bone remodeling with excessive bone resorption. Our previous data showed that matrix metalloproteinase 13 (MMP13) is highly expressed in MM cells, and co-culture with stromal cells further increased MMP13 levels. Most importantly, exogenous MMP13 increased OCL fusion and bone resorption activity induced by nuclear factor kappa-B ligand (RANKL). Here, we further addressed the mechanism of MMP13 upregulation in MM and its role in MM-related bone disease in vivo. Methods and Results: Based upon the previous results that IL-6 neutralizing antibody blocks MMP13 upregulation in MM cells co-cultured with stromal cells, we further investigated the role of IL-6 on MMP13 induction in MM cells. Human RPMI8266 MM cells were serum-starved for 24 hours, then treated with IL-6 for up to 96 hours. RT-PCR and western blotting showed that MMP13 expression and secretion by MM cells were upregulated after 24h and prolonged through 96h. AP-1 binding sites were identified in the MMP13 promoter, and we found that IL-6 induced upregulation of the AP-1 members c-Jun and c-fos in MM cells by RT-PCR and western blotting, which correlated with MMP13 induction. To address the role of heightened MMP13 expression/secretion of MM on OCL formation and development of lytic lesions, we silenced MMP13 expression in MM cells by lentiviral mediated shRNA transduction. 5TGM1 mouse MM cells were infected with the pKLO.1-puro-vector (EV) or pKLO.1-puro-sh-MMP13 (MMP13 Knock down [KD]) lentivirus particles and cells were selected by puromycin. MMP13 KD was confirmed by RT-PCR and western blotting. To investigate the effects of MMP13 KD on OCL formation, we co-cultured mouse bone marrow cells (BMC) with either 5TGM1-EV or 5TGM1-MMP13 KD MM cells using a transwell system to permit only soluble molecule exchange. Co-culture with 5TGM1-EV MM cells significantly (p Conclusion: Our results demonstrate that IL-6-induced high level of MMP13 in MM cells is essential for multiple myeloma tumor growth, OCL induction and development of lytic bone lesions. Disclosures: Lentzsch: Celgene: Consultancy, Research Funding.

Abstract 2908 Introduction: The eukaryotic initiation factor 4E (eIF4E) is a critical regulator in protein synthesis. It has been shown that overexpression and/or activation of eIF4E is critical for oncogenic protein synthesis. The precise role of protein translation in multiple myeloma (MM) is less clear. Recently it has been shown that eIF4E protein levels are higher in primary CD138+ MM cells than in normal plasma cells (Li, Jin et al. 2011) and that mutations in genes related to mRNA translation are involved in the pathogenesis of multiple (Chapman, Lawrence et al. 2011). Therefore, understanding the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. In this study, we analyzed the effects of introduction of ectopic eIF4E in MM cell lines compared with their parent cells in vitro and in vivo. Results: To examine the effect of overexpressed eIF4E in MM, we transduced MM cell lines with lentiviral particles encoding human eIF4E with GFP as selection marker. Introduction of ectopic eIF4E significantly increased critical factors for myeloma cell growth such as myc, cyclin D1, C/EBP beta and IRF4 as detected by western blotting. Overexpression of eIF4E resulted in a significant (p Conclusion: Here we show that eIF4E, a key player in translational machinery, promotes multiple myeloma cell growth both in vitro and in vivo. When eIF4E is overexpressed, it enhances protein expression of a subset of transcripts encoding regulators of the cell cycle and proliferation. Disclosures: Lentzsch: Celgene: Consultancy, Research Funding.

Abstract 1853 Methods: The translation initiation factor eIF4E is central to protein synthesis in general, and overexpression and/or activation of eIF4E is associated with a malignant phenotype by regulating oncogenic protein translation. Several previous publications indicate that aberrant control of protein synthesis contributes to lymphoma genesis but the exact role of protein translation in multiple myeloma (MM) is less clear. Therefore, understanding the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. The goal of this study was to determine the role and regulation of eIF4E, as well as the effects of protein translation controlling drugs in MM. Results: By western blot analysis as well as RT-PCR we found that eIF4E protein and mRNA levels are significantly elevated (up to 20 fold) in MM cell lines (H929, RPMI-8226, MM.1S and OPM2) and primary myeloma cells compared to normal plasma cells. Silencing of eIF4E gene expression in RPMI-8226 MM cells by a stable and inducible shRNA system significantly decreased viability of myeloma cells (by ∼ 43%) but not of HEK 293 suggesting a higher dependency of MM cells to protein translation. Next we evaluated different drugs including pomalidomide, rapamycin, pp242, 4EGI-1 and ribavirin, that are known to inhibit protein synthesis for their effects on protein translation in MM. By m7GTP pull down assays we evaluated the effects of the different drugs on eIF4E expression and activity. Rapamycin blocked the phosphorylation of 4EBP1 and eIF4E release, and subsequently inhibited eIF4G binding. The compound 4EGI-1 decreased the interaction between eIF4E and eIF4G. Pomalidomide decreased eIF4E protein expression. All drugs inhibited MM cell DNA synthesis measured by 3H-Thymidine incorporation. Treatment with pomalidomide (10uM), rapamycin (40nM), pp242 (10uM), 4EGI1 (50uM) or ribavirin (50uM) for 48h significantly decreased (p Conclusion: Here we show that eIF4E, a key player in translational control, is highly expressed in MM cells and critical for MM growth and survival. Therefore our study helps to understand the function and regulatory mechanism of eIF4E in MM. Further the evaluation of drugs targeting protein translation provides the basis for the optimization of current MM treatment or to open up new strategies such as targeting protein translation in future MM therapy. Disclosures: Lentzsch: Celgene Corp: Consultancy, Research Funding; Onyx: Consultancy; Genzyme: Consultancy; prIME Oncology: Honoraria; Imedex: Honoraria; Clinical Care Options: Honoraria.

Abstract 2973 We have recently shown that loss of STAT1 signaling in donor T cells significantly reduces acute GVHD in both MHC-mismatched and mHA-mismatched BMT and is associated with increased expansion of Treg cells. In this study we further addressed the role of STAT1 in GVL reactions and the underlying mechanisms. We first sought to delineate the role of STAT1 in the generation of CD8+ CTL against host-type spleen cells and A20 lymphoma targets using standard CML assays. Whole spleen cells (SPCs) or CD8+ selected STAT1+/+ or STAT1−/− cells from 129Sv mice (H2b) were stimulated for 5 days with irradiated BALB/c (H2d) spleen cells or A20 cells and then assessed for CTL activity against 51Cr-labelled target cells. STAT1-deficient SPCs appeared to have reduced CTL activity against host SPCs and host type A20 lymphoma cells compared to STAT1+/+ splenocytes. In contrast, isolated STAT1-deficient CD8+ cells had significantly increased CTL activity against both host-type targets and A20 cells. We then assessed the role of STAT1 in mediating GVL effects in vivo using a fully MHC-mismatched (129 [H2b]→BALB/c [H2d]) strain combination and the A20 lymphoma model. Lethally irradiated (800rad) BALB/c mice received T cell-depleted (TCD) wildtype 129 bone marrow cells (BMCs, 5×10E6) plus pan-T cells (5×10E5) from either 129.STAT1−/− or 129.STAT1+/+ donors. In addition, recipients were injected with 5×10E5 luciferase-expressing A20 lymphoma cells (A20-luc) on day 0. As expected animals receiving STAT1-deficient T cells showed significantly reduced GVHD-induced mortality (mean survival time [MST] not reached versus 11 days, p Disclosures: Lentzsch: Celgene: Consultancy, Research Funding; Onyx: Consultancy; Genzyme: Consultancy; Centocor Ortho Biotech: Research Funding.

Abstract 4026 Interferon-g/STAT1 signaling plays a critical role in regulating dendritic cell activation and function. Blockade of IFN-g signaling leads to reduced DC activation and impaired anti-tumor and acquired adaptive immunity. We recently reported that lack of IFN-g/STAT1 in donor lymphocytes leads to reduced GVHD induction in both MHC- and mHA-mismatched mouse BMT models. In this study, we addressed the role of host STAT1 in the regulation of GVHD. Wildtype or STAT1-deficient 129 mice (H2b) underwent allogeneic Bone Marrow Transplantation (BMT) following lethal irradiation (1044 rad). GVHD was induced using either BALB/c or B6 donor spleen cells. We unexpectedly observed that absence of STAT1 in recipient mice led to increased GVHD-associated mortality in both MHC-mismatched (MST 5 vs. 8, p=0.01) and mHA-mismatched (MST 11 vs. 23, p To determine the mechanism underlying the enhanced expansion of donor T cells in response to stimulation with STAT1-deficient APC, we hypothesized that STAT-deficiency may impair expression of the T cell inhibitory molecules Programed Cell Death-Ligand1 or-2 (PD-L1,-L2) on APC. We therefore studied the expression of PD-L1 and PD-L2 expression on wildtype and STAT1-deficient DC. Indeed, were able to demonstrate that absence of STAT1 significantly suppressed PD-L1 expression on BMDCs upon in vitro LPS stimulation (Mean Fluorescence Intensity 167.2± 15.9 vs. 532.5±7.6, p Disclosures: Lentzsch: Centocor Ortho Biotech: Research Funding; Genzyme: Consultancy; Onyx: Consultancy; Celgene: Consultancy, Research Funding.

Abstract 1840 Introduction: The IMiD® immunomodulatory drugs lenalidomide (LEN) and pomalidomide (POM) yield high response rates in patients with multiple myeloma (MM). However, the use of IMiDs is associated with neutropenia and thrombocytopenia. It has been shown before that IMiDs down-regulate the transcription factor PU.1/SPI1 leading to maturational arrest of granulocytes with accumulation of immature myeloid precursors and subsequent neutropenia (Blood. 2010; 115:605-614). However, the mechanism underlying the development of thrombocytopenia is unclear. Methods and Results: Here, we investigated the effects of IMiDs on megakaryopoiesis. In megakaryocytic colony formation assays IMiDs significantly (p The finding that IMiDs induce development of megakaryocytic precursors is in contrast to the clinical observation that IMiDs induce thrombocytopenia. Since previous studies showed that IMiDs are not directly toxic to bone marrow hematopoietic cells (Blood. 2005; 105:3833-40), we hypothesized that these effects might be induced by a maturational arrest of megakaryopoiesis resulting in thrombocytopenia. Therefore we studied the morphology of Mks for signs of immaturity. Giemsa staining and CD61 immunohistochemistry showed Mks developed in the presence of IMiDs were smaller in size with hypo-lobulated nuclei. 45% of cells treated with LEN, and 52% treated with POM exhibited a single nuclear lobe compared to 23% of cells treated with DMSO. We confirmed our findings by measuring the ploidy of cultured Mks by FISH. Mks (CD61-FITC-positive) were studied from each treatment (DMSO and IMiDs) and the number of nuclei and the number of centromeric region of chromosome 6 (CEP 6) signals for each Mk were recorded. CD61-FITC-negative cells were omitted from the FISH analyses. The percentage of 2N (disomic) cells was 2.5-fold higher (p=0.0221) in the IMiDs group compared to control group; in contrast the percentage of 4N and ≥8N cells (with multiple disomic nuclei) decreased 1.5 and 1.3-fold (p Since failure of terminal differentiation and excessive proliferation of Mks has been described in GATA1-deficient Mks (Blood. 2005; 106:1223-1231) we analyzed the expression of GATA1 at the mRNA and protein levels after treatment with IMiDs using real-time PCR, Western blot and immunofluorescence microscopy, respectively. IMiDs-treated Mks showed down-regulation of GATA1 associated with decreased expression of FOG1/ZFPM1 and NFE2 both critically interacting with GATA1. Further, previous studies suggested that polyploidy formation in Mks depends on the expression of cyclin D1 isotypes (Blood. 2007; 109:5199-5207). Indeed, IMiDs treatment caused a decrease of p21 and CCND1. Conclusion: Taken together, our findings suggest that IMiDs maintain and expand early hematopoietic progenitors up to several weeks. Our data further indicate that the loss of key transcription factors such as GATA1 and CCND1 both critical for megakaryopoiesis precludes Mks from continued maturation. This might lead to maturational arrest of Mks with accumulation of immature megakaryocytic precursors and subsequent thrombocytopenia. Disclosures: Lentzsch: Celgene Corp: Consultancy, Research Funding; Onyx: Consultancy; Genzyme: Consultancy; prIME Oncology: Honoraria; Imedex: Honoraria; Clinical Care Options: Honoraria.

Abstract 135 Background: Lenalidomide and pomalidomide are IMiD® immunomodulatory compounds that have been shown to be highly active in the treatment of multiple myeloma (MM). IMiD compounds exert their anti-tumor effects via acting on costimulatory proteins of T cells and NK cells, augmenting both the adaptive and innate immune system. But the mechanisms by which IMiD compounds directly inhibit MM cell proliferation are still unclear. Here we focused on the direct effects of IMiD compounds alone on MM cells. Results and Methods: We found that IMiDs, at concentrations as low as 0.01 μ M, induce significant inhibition of DNA synthesis in MM cells as shown by thymidine uptake. Since our previous work demonstrated that C/EBPβ is an important transcription factor which controls the growth and proliferation of myeloma cells, we analyzed the effects of IMiD compounds on C/EBPβ. We found that both pomalidomide and lenalidomide significantly decreased the protein level of C/EBPβ LAP-isoform in MM cell lines and primary MM cells. IMiD compound-induced suppression of C/EBPβ protein expression led to impaired transcription of the downstream IRF4, and subsequently to downregulation of BLIMP1 and XBP1, which are all critical for MM survival. To confirm our findings in vivo, we analyzed IRF4 expression by double labeling (IRF4+/CD138+) immunohistochemical staining of bone marrow biopsy samples of 23 myeloma patients prior to therapy and during therapy with lenalidomide. During lenalidomide therapy, the bone marrow MM cells showed a significantly weaker staining intensity for IRF4 in comparison to prior therapy. This was quantified by a significant (p Conclusions: Our studies, for the first time, provide evidence that IMiD compounds inhibit MM cell proliferation and survival by affecting the translation of C/EBPβ and subsequently multiple downstream transcription factors including IRF4, BLIMP1 and XBP1. Due to the critical role of C/EBPβ in mediating effects of IMiD compounds in MM, it might be a target to overcome drug resistance to IMiD compounds. The fact that pomalidomide can overcome resistance to lenalidomide in MM requires still further evaluation. Disclosures: Schafer: Celgene Corporation: Employment, Equity Ownership. Mapara:Gentium: Equity Ownership. Lentzsch:Celgene Corp: Research Funding.

Abstract 4079 Background: In multiple myeloma (MM), lenalidomide has impressive clinical activity in patients with both relapsed/refractory and newly diagnosed disease. Nevertheless hematopoietic stem cell transplantation (HSCT) is still the “backbone” in the treatment of newly diagnosed MM patients and very often lenalidomide treatment (as induction therapy) is combined with HSCT in order to achieve high response rates. Clinical data have shown that in up to 43% of those patients, standard mobilization of CD34+ cells with G-CSF alone failed to mobilize significant numbers of hematopoietic progenitors into peripheral blood, raising concerns about potential stem cell toxicity of lenalidomide (Mazumder et al, Leukemia 2008). Interestingly mobilization of hematopoietic progenitors with AMD-3100 (Plerixafor) overcomes mobilization failures in almost all patients previously treated with lenalidomide. The fact that AMD-3100 antagonizes the binding of chemokine stromal- cell–derived factor-1α (SDF-1α) to CXC chemokine receptor 4 (CXCR4) suggests a potential role of the CXCR4/SDF-1α axis in mediating mobilization failure after lenalidomide treatment. Subsequently, the findings noted above raised questions on: 1) the stem cell toxicity of lenalidomide; 2) the underlying mechanism that mediates the development of G-CSF resistance; and 3) the mechanism of the modulation of the CXCR4/SDF-1α axis by lenalidomide. In our previous work we showed the following. 1) Lenalidomide neither inhibited colony formation in standard colony assays nor the development of cobble stone area forming cells (CAFC) in LTC-IC assays, suggesting that lenalidomide is not stem cell toxic (Koh et al, Blood 2005). 2) We further showed that lenalidomide significantly upregulated G-CSF secretion of CD34+ cells (600%), suggesting that the high levels of G-CSF may mediate a relative resistance towards G-CSF-induced mobilization (Pal et al, Blood, 2010). Results and Methods: We analyzed why blocking CXCR4 by AMD-3100 overcomes mobilization failure to G-CSF. We first examined the CXCR4 expression profile of CD34+ cells treated with lenalidomide. Lenalidomide treatment significantly upregulated the expression of CXCR4 on cell surface after 48h treatment measured by flow cytometry. Increased expression of CXCR4 onCD34+ cells remained high with continuous treatment. Western blot assay of the hydrophobic (membrane) and the hydrophilic (cytosol) cell fraction confirmed our data showing that lenalidomide increases the expression of CXCR4 on CD34+ cell surface. Confocal microscopy showed that lenalidomide inhibited SDF-1α induced CXCR4 internalization. In accordance with the increased CXCR4 surface expression, transwell migration assay revealed that the SDF-1α-induced migration of CD34+ cells in the presence of lenalidomide significantly increased by 52% in comparison to control. Quantitation of SDF-1α showed that CXCR4 had no significant effect on the secretion of SDF-1α by CD34+ cells and in human stromal cells. Result: In conclusion, our data show that lenalidomide is not toxic to hematopoietic progenitors. The strong increase of G-CSF secretion by CD34+ cells might contribute to a desensitization of CD34+ cells to G-CSF mobilization. Primarily our data indicate that increased expression of CXCR4 followed by blocked internalization, increases binding to SDF-1α secreted by the bone marrow niche. This subsequently prohibits the mobilization of CD34+ cells. These data suggest that blocking the CXCR4 receptor by AMD-3100 disrupts this circle and finally permits the mobilization of hematopoietic cells from the bone marrow niche into peripheral blood. This study provides novel insights into the effects of lenalidomide on CD34+ cells relevant for HSCT in MM. Disclosures: Roodman: Amgen: Consultancy; Celgene Corp: Consultancy; Acceleron: Consultancy; Millennium: Consultancy. Mapara:Gentium: Equity Ownership. Lentzsch:Celgene Corp: Research Funding.

Abstract 1844 Poster Board I-870 Background: Lenalidomide is an immunomodulatory drug that has been shown to be highly active in the treatment of multiple myeloma (MM). In combination with dexamethasone, remission rates of up to 61% are achieved in newly diagnosed MM patients. Nevertheless, the mechanisms of how immunomodulatory compounds induce high remission rates in MM remains elusive. Methods and Results: Here we investigate the underlying anti-MM mechanism of lenalidomide. Lenalidomide significantly (p Disclosures: Schafer: Celgene Corporation: Employment. Lentzsch:Celgene: Consultancy, Speakers Bureau; Pfizer: Consultancy.

Abstract 296 Background: Benzimidazoles, originally categorized as broad-spectrum anthelmintic drugs, have been recently reported to induce growth arrest and apoptosis in some solid cancer models (e.g. colorectal and lung). We performed a multiplex drug-screening assay that identified benzimidazoles as potential anti-multiple myeloma (MM) agents. Methods and Results: In this study, we demonstrate that one of the benzimidazole members, nocodazole, inhibited proliferation and induced apoptosis in MM cell lines and primary MM cells alone and in co-culture with bone marrow stromal cells. The resistant phenotype of those MM cells resistant to conventional therapies could be completely reversed by nocodazole. The IC50 values were 60 nM (RPMI8226-S), 25 nM (RPMI8226-Dox40), 80 nM (RPMI8226-MR20), 60 nM (RPMI8226-LR5), 65 nM (MM.1S) and 60 nM (MM.1R). Viability of primary cells decreased by 66% in CD138+ cells and 7% in CD138− mononucleated bone marrow cells after 48 hour treatment. Cell cycle analysis revealed a G2/M arrest and subsequent cell death induced by nocodazole. Nocodazole also caused morphologic elongation in MM cells in a dose-dependent manner during prometaphase. The morphologically changed cells exhibited a microtubular network disarray as evidenced by microtubular immunofluorescent staining. Signaling studies indicated that increased expression of Bim protein and reduced XIAP and Mcl-1 levels were involved in nocodazole-induced apoptosis. Further investigation showed Bcl-2 phosphorylation as a critical mediator of cell death, which was triggered by the activation of JNK, instead of p38 kinase or ERKs. Treatment with JNK inhibitor SP600125 completely inhibited Bcl-2 phosphorylation at Ser70 and Thr56 induced by nocodazole. Nocodazole-induced cell death subsequently decreased from 79% to 28% after pretreatment with SP600125. Combination of nocodazole with dexamethasone induced significantly stronger induction of cell death at either drug dose. Dexamethasone at 20 nM, nocodazole at 15 or 30 nM could only induce 19%, 10.3% and 16% cell death, respectively. However, their combinations resulted in 67% and 92% nuclear fragmentation, respectively. Based on our in vitro data, we analyzed nocodazole in a SCID xenograft murine model. Nocodazole alone (5 and 20 mg/kg) or combined with dexamethasone (2 mg/kg) at a lower dose of 12 mg/kg significantly inhibited H929 tumor growth and prolonged survival in a SCID xenograft murine model. Conclusions: Our studies demonstrate that nocodazole has a potent anti-MM activity and might be a promising new treatment approach for MM. * Supported by a grant from the Multiple Myeloma Research Foundation. Disclosures: Roodman: Acceleron: Consultancy; Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy. Lentzsch:Celgene: Consultancy, Speakers Bureau; Pfizer: Consultancy.