Citations

Abstract Activation of NFkB has been noted in late B-cell malignancies, but no genetic basis for this activation has been shown except for rare mutations of the NFkB pathway in lymphoma and none have been described in MM. Bortezomib, highly tumoricidal in a subset of MM patients, inhibits NFkB suggesting it’s role as a therapeutic target. Here we show for the first time the critical importance of mutations that dysregulate this potential survival pathway in human myeloma cell lines (HMCL) and new diagnosis MM patients. We performed high-density oligonucleotide array CGH (Agilent 44k) on CD138+ selected MM cells from 68 patients and 42 unique HMCL. Initially we focused on small loci, bi-allelically deleted, as markers for inactivation of potential tumor suppressor genes. We next developed FISH probes for the identified loci to confirm the bi-allelic deletions seen by aCGH. We next sequenced all samples with monoallelic deletions, and additional unselected patients, and identified additional inactivating mutations. Multiple novel tumor suppressor genes (TSG) were identified and validated, whose loss of function (both in patients and HMCL) leads to activation of the non-canonical NFkB pathway including: TRAF2 (9q34), TRAF3 (14q32), BIRC2/BIRC3 (11q22) and CYLD (16q12). Functional studies showed that HMCL with deletion or mutations in these genes have markedly increased NFkB2 p52/p100 ratios relative to other HMCL (and for BIRC2/BIRC3 relative to an isogenic line that retains one copy of BIRC2/BIRC3), indicating genetic activation of the non-canonical NFkB pathway. Reintroduction of deleted TSG into HMCL inhibited cell growth and resulted in cell death (see data on abstract by J. Keats et al). We next focused our analysis (of combined aCGH and GEP data) on positive regulators of this pathway. Rare IgH translocations of NIK have recently been identified in MM. By aCGH we identified amplification and rearrangements of NIK. We identified dysregulated expression of TNF family receptors that activate the non-canonical pathway: CD40 (translocation), TACI (amplification) and LTBR. All of the HMCL with these mutations also showed increased NFkB2 p52/p100 ratios, consistent with activation of the non-canonical NFkB. These mutations correlate with a gene expression profile of NFkB activation that is detected in one half of patients. In summary, we have determined for the first time the genetic basis for constitutive NFkB activation - present in 19/42 HMCL, and we estimate in at least one quarter of newly diagnosed patients (approximately one half of those with evidence of NFkB activation by GEP). We propose a mechanism in which positive (TACI, CD40, LTBR) and negative regulators (TRAF2, TRAF3, BIRC2/BIRC3, CYLD) of the pathway converge on NIK to mediate the processing of NFKB2 p100 to p52. The preponderance of genetic evidence suggests that drugs that target the non-canonical NFkB pathway, will be most effective in treating the 50% of MM patients with NFkB activation.

Abstract From a high throughput RNAi screen of the human druggable genome targeting the KMS11 cell line, we identified the suppression of the vacuolar H+- ATPase (V-ATPase) family as cytotoxic to myeloma cells. In the screen, two oligos against each gene for the V-ATPase subunits ATP6V1A and ATP6V1B1 resulted in suppression of cell growth (50% and 60% inhibition of cell viability respectively). We further confirmed this result using both lentiviral shRNA knockdown and two small molecule inhibitors specific for V-ATPase. Silencing of ATP6V1A by lentiviral shRNA knock-down in KMS11 and in OPM1 myeloma cell lines caused 75–80% reduction of cell viability at 5 days post infection (measured by MTT assay). Consistent with this result, the V-ATPase specific inhibitors, bafilomycin A1 and REATA 203, both inhibited the growth of a genetically heterogeneous and standardized panel of 14 human myeloma cell lines in vitro with an IC50 ranging from 2.2 – 8.9 nM (mean 5.25 nM) for bafilomycin A1 and 46–1594 nM (mean 542.5 nM)) for REATA 203. We further demonstrated that patient samples (n=10) were sensitive to 20nM bafilomycin A1 which induced a mean of 58% of MM cells to undergo apoptosis (range 10% to 93%) after 24 hours of treatment. Similar to bafilomycin A1, treatment of primary patient-derived MM cells with 500 nM REATA 203 for 72 hours resulted in a mean 69% apoptosis (range 24% to 97%). In contrast, non-myeloma cells (the CD138- fractions of the bone marrow samples) were less sensitive - mean 9% apoptosis (range from 0% to 34%) under the same treatment conditions. Of high interest, however, unlike most drugs we have studied in pre-clinical myeloma models, the cytotoxicity induced by bafilomycin A1 in MM cell lines is abrogated by co-culture with patient bone marrow stromal cells but is not affected by IL-6 or IGF-1 treatment. Dexamethasone- or melphalan-resistant MM cell lines were also highly sensitive to both bafilomycin A1 and REATA 203. In a xenographic JJN3 mouse model, bafilomycin A1 suppresses and delays growth of tumor in a dose-dependent fashion. Gene expression analysis of normal-donor bone marrow plasma cells (n=19), primary tumor samples from MM patients (n=107) and normal somatic tissues demonstrates ubiquitous expression of most subunits of V-ATPase, however, some subunits are preferentially expressed in myeloma cells compared with normal plasma cells, including ATP6V1F (84% vs. 11%), ATP6V1E1 (29% vs. 5%), ATP6V1G2 (17% vs. 0%) and ATP6V0E 2 (36% vs. 16%). In conclusion, our data indicate that vacuolar H+-ATPase inhibitors are of interest as potential therapeutics for MM.

Abstract Bortezomib and related proteasome inhibitors have profound pre-clinical and clinical activity in Multiple Myeloma (MM). However, the critical molecular target(s) modulated by proteosome inhibition remains unknown. The overall response rate to bortezomib in relapsed and refractory MM patients is consistently 35%–50%, suggesting specific molecular targets or resistance mechanisms are inherent in patients. None of the known effects of bortezomib has been shown to correlate with clinical responses in patients or to explain the unique sensitivity of MM (as opposed to other malignancies) to these reagents. Therefore, in this study, we have conducted a small interfering RNA (siRNA) screen of the druggable genome in human myeloma cells to identify genes that modulate sensitivity to bortezomib. KMS11 human myeloma cells were reverse transfected with 13,984 synthetic short interfering RNAs (siRNA) against each of 6992 druggable targets (2 siRNA/gene with a single-siRNA per well/384 well format). At 24 hours post transfection, bortezomib was added at IC10, 30, 60 and 90. Cell viability was measured by ATP-dependent luminescence assay 72 hours post drug treatment. The resulting data from primary screening was evaluated for multiple quality control metrics and found to exceed all expected performance parameters with >99% global transfection efficiency, <0.25 CV values across the dataset with minimal plate-to-plate and set-to-set variation observed. From analysis of the primary screening data, a core panel of 320 (4.6% of druggable targets in library) bortezomib sensitizers were identified. 187 (58%) of these were further confirmed in a secondary screening experiment by multiple (2 to 4) independent siRNA. Among these 187 genes (2.6% of the genes investigated), some known targets, such as proteasome subunits are evident. Other genes belonging to the same functional gene families (e.g. TNF receptor family, Ras family, kinases, oncogenes and transporter proteins) or functioning within the same network of signal transduction (e.g. NF-kB, Wnt and estrogen receptor pathways) are evident. Our primary screen also identified genes that attenuate bortezomib-induced cytotoxicity. Members in this group include genes implicated in the negative regulation of cell proliferation and survival, such as NF-kB inhibitors, JNK pathway activators, tumor suppressors, apoptosis inducers and phosphatases. These studies enable dissection of the molecular mechanism of bortezomib in myeloma cells and may serve to point to biomarkers for response prediction and novel chemotherapeutic combination therapies.

Introduction We have shown that in myeloma (MM) the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide, in a CRBN dependent fashion, inhibit thioredoxin reductase and thus increase intracellular oxidative stress as a consequence of peroxide accumulation (Sebastian et al. 2017). Accordingly, we have also shown that CRBN expression alone does not fully correlate with lenalidomide sensitivity, as the cells ability to decompose H2O2 is important - MM cells with lower H2O2 decomposition capacity are more sensitive to IMiDs. We hypothezised that the specific metabolic pathways used by cells for biomass production would influence IMiD sensitivity since they also influence this cellular oxidative capacity. Cellular proteins are key elements of this biomass, and required for the proliferation of MM cells and production of immunoglobulin proteins. Methods We cultured MM cells under various nutrients conditions to understand its effect on cell proliferation and drug responses. Cell viability was assessed by MTT assay, the Luminescent Assay, and direct cell counting. MM cells glycolytic rate was measured using the Seahorse XF analyzer. Western blots were performed to quantify various protein expression levels in MM cells cultured under different nutrients conditions. CRBN CRISPR/Cas9 KO plasmid was used to generate CRBN knockout cells and a lentivirus system was used to over-express CRBN in MM cells. Results MM cells resistant to lenalidomide, but still expressing functional CRBN, show a higher glycolytic pathway use than sensitive cells. We found that the lenalidomide sensitive cell line MM1.S is highly dependent on extracellular glutamine for cell proliferation. In contrast, JJN3, a lenalidomide resistant cells line with wild type CRBN, preferentially consumes glucose. We found that glutamine depletion from culture media completely abolished lenalidomide sensitivity in MM1.S cell line. Western blot analysis revealed that antibody production by MM1.S was primarily dependent on glutamine availability, and that JJN3 antibody production was dependent upon glucose availability. While glutamine depletion from culture medium completely abolished lenalidomide sensitivity in MM1.S cell line, IKZF1, and IKZF3 degradation was unchanged. We further studied the role of CRBN in glutamine dependent cellular biomass protein production using isogenic cell lines (+/- wtCRBN). We found that wtCRBN expressing cells proliferate more and show higher antibody production in the presence of glutamine, over CRBN negative isogenic cell lines. Lenalidomide treatment further increased glutamine dependent antibody production in wtCRBN expressing cells. One possibile explanation is that activation of glutamine catabolism can facilitate protein demethylation via the supplementation α-ketoglutarate (α-KG). Therefore, we tested whether lenalidomide treatment could induce protein demethylation in sensitive cell lines. Western blot probed with mono-methyl lysine antibody showed lenalidomide induced protein demethylation. We further confirmed that protein demethylation, and lenalidomide sensitivity is a consequence of elevated α-KG by treating cells with cell-permeable 5-octyl- α-ketoglutarate which act as a substrate of the lysine demethylases. 5-Octyl-α-ketoglutarate treatment inhibited cell proliferation preferentially in wtCRBN expressing cells, and also enhanced lenalidomide induced sensitivity. Protein demethylation is associated with protein ubiquitination and proteasomal degradation. We thus hypothesize that lenalidomide induced protein demethylation also likely increases proteasome inhibitors sensitivity in MM. Conclusion MM cells with preferential glutamine consumption are likely to be more sensitive to lenalidomide, and extracellular glutamine depletion can induce lenalidomide resistance. MM cells expressing CRBN, but not dependent on glutamine for protein biomass production are more likely to be resistant to lenalidomide. Our study postulates that CRBN protein requirement in lenalidomide sensitivity cells is restricted to high glutamine dependency, and that it is quite likely CRBN has a role in glutamine metabolism. Moreover, MM cells that consume more glutamine are under higher oxidative stress and exhibit less H2O2 decomposition capacity and therefore increasing sensitivity to IMiDs. References Sebastian , S., et al., Blood 2017 129:991-1007 Disclosures Stewart: Ono: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Bristol Myers-Squibb: Consultancy; Celgene: Consultancy, Research Funding; Ionis: Consultancy; Janssen: Consultancy, Research Funding; Oncopeptides: Consultancy. Bergsagel:Janssen Pharmaceuticals: Consultancy; Ionis Pharmaceuticals: Consultancy; Celgene: Consultancy. Fonseca:AbbVie, Amgen, Bayer, Celgene, Kite, Janssen, Juno, Merck, Pharmacylics, Sanofi, Takeda: Other: Consultant/Advisor; Prognosticatin of MM based on Genetic Categorization by FISH: Patents & Royalties; Adaptive Biotechnologies: Other: Scientific Advisory Board.

Background Bortezomib (BTZ) is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. The underlying mechanisms of such proteasome inhibitor resistance are still incompletely understood. Methods To further understand its resistant mechanism, we generated eight multiple myeloma (MM) cell lines resistant to bortezomib (BTZ) by exposure to increasing drug concentration: five of them acquired novel PSMB5 mutations. Given the rarity of similar mutations in over 1,500 analyzed MM patients, we explored in depth the role of the proteasome on MM cell viability and BTZ sensitivity by systematically deleting the major proteasome targets of BTZ by CRISPR. Results We demonstrated that MM cell lines without PSMB5 were surprisingly viable (mutation corresponding yeast gene pre2 is lethal). PSMB5 mutated, BTZ resistant, MM cell lines were re-sensitized to BTZ when PSMB5 was experimentally deleted, implying that this mutation is activating in its drug resistance function. In contrast PSMB6 knockout was lethal to MM cell lines, which were efficiently rescued by re-introduction of wild type PSMB6. Interestingly, reduction in PSMB6 levels also prevented the splicing of the major catalytic subunits PSMB5, PSMB7, PSMB8 and PSMB10. PSMB6 engineered with no splicing function or catalytic activity, also restored viability, inferring that the contribution of PSMB6 to proteasome structure is more important than functional activity. Supporting this observation, BTZ sensitivity was restored in resistant MM cells line by introducing low level expression of mutated PSMB6 lacking splicing function. As with PSMB6, PSMB7 knockout was lethal to MM cell lines. In contrast, loss of immunoproteasome subunits PSMB8 and PSMB9 was neither lethal nor restored sensitivity to BTZ. Our results demonstrate that expression of the three constitutive proteasome subunits PSMB5, PSMB6 and PSMB7 is highly co-dependent. This dependence is relying on the structure, but not the function, of PSMB5 and PSMB6. Conclusions In summary, PSMB5 and PSMB6, but not PSMB8 and PSMB9, are highly relevant for BTZ sensitivity in MM. Absence of PSMB6 or PSMB7, but not PSMB5, was lethal in MM cell lines. Expression of PSMB5, PSMB6 and PSMB7 was highly co-dependent. Together these findings suggest that the modulation of expression rather than function of PSMB5, PSMB6 or PSMB7 may be a new therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

Abstract Immunomodulatory drugs (IMiDs) are widely used in the treatment of patients with Multiple Myeloma (MM) however, only 30% of relapsed MM patients respond to single agent therapy and most patients eventually develop drug resistance. The molecular target of IMiDs in MM is cereblon but other parallel pathways or downstream events which enhance or preclude drug responsiveness are unknown. We therefore conducted a genome scale small interfering RNA (siRNA) lethality study in MM in the presence of increasing concentrations of lenalidomide. Primary screening was performed in a single-siRNA-per-well format with the human druggable genome siRNA set V4 comprising four siRNAs targeting each of 6,992 genes (total 27968 siRNAs). Lenalidomide was added 24 hours post transfection and cell viability was measured by ATP-dependent luminescence at 144 hours after transfection. Primary screen data was rigorously evaluated for multiple quality control metrics and found to exceed all expected performance parameters with >98% global transfection efficiency, <0.25 CV values, and minimal plate-to-plate and set-to-set variations observed. Hit selection was performed by analysis of IC50 value shift in the presence of each testing siRNA compared with three different control siRNA oligos. 160 candidate genes that enhance lenalidomide sensitivity upon silencing (sensitizers) were selected and re-screened with four siRNA oligos targeting each gene. 50 genes were identified as reproducible lenalidomide sensitizers including three Peroxisome (PEX) family proteins (PEX1, PEX10 and PEX7) and seven RAB family proteins (RAB17, RAB1A, RAB26, RAB30, RAB36, RAB4A and RAB8A). Four kinase genes were also identified in sensitizer hits and two of these, I-Kappa-B Kinase-Alpha (IKK1 or CHUK) and ribosomal protein S6 kinase (RPS6KA3 or RSK2), encode proteins that associate with significance together with a phosphorylation dependent transcription factor (CREB1) in Toll signaling pathways (p-value 0.0068). RSK2 is a serine/threonine-protein kinase that acts downstream of oncogenic FGFR3 mediated signaling and is phosphorylated by ERK (MAPK1/ERK2 and MAPK3/ERK1 signaling) during hematopoietic transformation. Phosphorylated RSK2 was previously reported to be frequently expressed in myeloma cell lines and primary myeloma cells. Using lentiviral shRNA expression, we demonstrated that knockdown of RSK2 in three genetically variable MM cell lines induced cyctocytoxiticy and consistently sensitized to lenalidomide. Two selective small molecular inhibitors of RSK2 (SL 0101-1 and BI-D1870) were then demonstrated to synergize with lenalidomide to induce myeloma cell cytotoxicixity. To further understand the mechanism underlying sensitization, immunoblotting analysis was performed to look at downstream changes after either RSK2 knockdown or RSK2 inhibition by BI-D1870. We found that both RSK2 knockdown and BI-D1870 treatment, mimicking lenalidomide treatment or cereblon inhibition, induced downregulation of both IRF4 and MYC in MM cells. The combination of lenalidomide and BI-D1870 not only produced a substantial synergistic effect inducing MM cytotoxicity, but also demonstrated a significant enhancement of downregulation of IRF4 and MYC. Forced overexpression of RSK2 attenuated the synergistic effects of lenalidomide and BI-D1870. In summary, our high throughput screen identified multiple gene targets that associate with increasing sensitivity to IMiDs in MM cells, of which, RSK2 was further validated by both shRNA silencing and specific inhibitors as an effective target to cooperate with IMiDs to induce myeloma cytotoxicity. Clinical studies of RSK2 inhibition in concert with IMiD (cereblon inhibitor) therapy would be appropriate. Disclosures: Stewart: Onyx: Consultancy, Research Funding; Millennium: Honoraria, Research Funding; Celgene: Honoraria; BMS: Honoraria.

Abstract As multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that inhibit signaling pathways driving cyclin D2 promoter transactivation, assaying more than 4,000 compounds. The top-ranked compound from these studies was a natural triterpenoid, pristimerin. Pristimerin markedly suppressed cyclin D2 promoter activity (>90%) in 3T3 fibroblast cells and inhibited cyclin D1, D2 and D3 protein expression in myeloma tumor cells. Strikingly, the early (4 hour) transcriptional response of myeloma cells treated with pristimerin closely resembles cellular responses elicited by proteosome inhibitors (P<10−9) (Connectivity Map Build 2, www.broad.mit.edu/cmap), with rapid induction of heat shock proteins (HSP70 >90-fold), activating transcription factor (ATF) 3 and CHOP. Enzymatic assays performed with purified 20S proteosome, or with total cellular extract, confirm that pristimerin rapidly and specifically inhibits chymotrypsin-like 20S proteosome activity at low concentration (<100nM), causing sustained inhibition lasting >6 hours. Consistent with inhibition of proteosome function, pristimerin causes rapid and sustained accumulation of high molecular weight poly-ubiquitinated protein in myeloma cell lines. Notably, related cytotoxic triterpenoid drugs, such as the methyl ester of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me, RTA 402) or betulinic acid or the ginsenosides - all of which show promising anti-cancer activities and are currently in clinical trials for advanced lymphoma, leukemia or solid malignancies - commonly inhibit NF-kB activation via direct inhibition of IKKα or IKKβ. In contrast drugs that function as proteosome inhibitors also commonly suppress NF-kB function instead by impairing degradation of ubiquitinated IkB. Immunoblotting for phosphorylated IkB confirms that pristimerin, like other triterpenoids, acts upstream of IkB to inhibit its phosphorylation, although pristimerin simultaneously inhibits proteosome activity with marked potency to diminish the clearance of ubiquitinated IkB. As a result of this two-fold stabilization of IkB, pristimerin causes overt and specific suppression of NF-kB mediated transcription, measured by a panel of transcriptional reporters with synthetic promoters containing 5x repeats of generic binding sites for NF-kB, AP-1, CREB or TCF4. Importantly, specific suppression of constitutive NF-kB transcriptional activity was pronounced in myeloma cells with inherent NF-kB pathway activation resulting from bi-allelic deletion of the TRAF3 tumor suppressor. Constitutive activation of the NF-kB pathway occurs in a significant proportion of primary myeloma tumors, most commonly via inactivation of TRAF3. Selective silencing of NF-kB driven transcription in myeloma cells may mediate the potent suppression of cyclin D proteins induced by this compound. Significantly, multiple myeloma cells are exquisitely sensitive to both proteosome inhibition or NFkB pathway inhibition. Consistent with these twin vulnerabilities, pristimerin is potently and selectively lethal to primary myeloma cells from patients (IC50<100nM) grown in mixed lineage culture and inhibits the growth of xenografted human plasmacytoma tumors grown in mice, providing a strong rationale for pharmaceutical development of triterpenoid dual-function proteosome-plus-NF-kB pathway inhibitors as therapeutics for multiple myeloma and related human malignancies.

Background The precise molecular mechanism and targets of lenalidomide specific anti-Multiple Myeloma (MM) activity remain largely unknown, although, cereblon (CRBN) has been identified as a central mediator of activity with IRF-4 acting as a downstream molecule. Lenalidomide resistance in MM cells which, despite CRBN depletion, are able to restore their IRF-4 levels suggests the existence of alternative pathways. Methods To measure apoptosis and gene expression analysis 1x 106cells were incubated with 10μm to 50μm of lenalidomide for 24 to 96 hours before they were examined by annexin-PI and FACS analysis. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. For lenalidomide followed bortezomib serial studies, cells were incubated with 10μm of lenalidomide for 48 hours and later treated with bortezomib for 24 to 48 hrs. CRBN and immunoglobulin light chain knock-down were performed by lentiviral mediated shRNA. Results We found lenalidomide induces Endoplasmic reticulum stress (ERS) via accumulation of intracellular immunoglobulin proteins, but only in the presence of CRBN in a panel of MM cell lines. This accumulation of intracellular proteins resulted in ERS as documented by western blot and RT-PCR in OPM2 and MMIS cell lines. We next utilized OPM2 cells knocked down for CRBN and control, non-target (NT), and treated with different doses of lenalidomide. After three days of treatment an ERS response was occurring in OPM2-NT cells but not in CRBN knockdown cells. Using Western blot we found decreased XBP-1u and increased XBP-1s, ERS marker proteins, in CRBN positive (NT) cells after 72 hours of lenalidomide treatment, but not in CRBN knock-down cells. Another ERS marker GRP78/BiP was also clearly accumulated after lenalidomide treatment in CRBN positive cells. Inactivation of the tumor suppressor p53 by degradation is a mechanism utilized by cells to adapt to ERS has been reported. We also found that p53 protein down regulated after lenalidomide treatment in CRBN positive OPM2 and MMIS cells. We have analyzed other isogenic cell lines MM1.S (lenalidomide-sensitive) and isogenic MM1.Sres (lenalidomide-resistant) HMCLs and found that lenalidomide induces ERS in MM1.S but not MM1.Sres cells. CRBN is important for lenalidomide to induce immunoglobulin protein accumulation and ERS was further proved by over-expressing CRBN in OCI-My5 MM cell line and subsequent lenalidomide treatment. To test the effect of light chain accumulation as ER stressors, we knocked down immunoglobulin light chain lambda in human MM cell line OPM2. Knock-down of lambda light chain in OPM2 cells resulted in lenalidomide resistance and it induced endoplasmic reticulum stress mediated anti-myeloma activity. Our preliminary data reveals that lenalidomide mediated progressive ERS can positively enhance bortezomib induced apoptosis in an in-vitro MM model. We pre-treated myeloma cell lines with lenalidomide and subsequently treated with bortezomib. MM cells pre-treated with lenalidomide for two days and there after treated with bortezomib clearly shown increased sensitivity to bortezomib induced apoptosis than non-pre-lenalidomide treated cells. Conclusion Here we show that lenalidomide causes accumulation of immunoglobulin light chains, inducing progressive ER stress, resulting in anti-myeloma activity and that effect depends on the presence of CRBN. Proteasome inhibitors such as bortezomib also sensitize MM cells to endoplasmic reticulum stress-mediated apoptosis. Our data demonstrated the synergistic effect of lenalidomide and proteasome inhibitors, likely due to the augmented protein stress. This data support the use of serial therapy utilizing pre-lenalidomide treatment followed by bortezomib or carfilzomib treatment. Disclosures: Fonseca: Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy, Research Funding; Binding Site: Consultancy; Millennium: Consultancy; AMGEN: Consultancy; Cylene: Research Funding; Prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties.

Abstract Abstract 602 The molecular target(s) which co-operate with proteasome inhibition in inducing drug sensitivity or resistance in Multiple Myeloma (MM) remain unknown. We therefore conducted a genome scale small interfering RNA (siRNA) lethality study in KMS11 MM cells in the presence or absence of bortezomib without regard to pre-conceived mechanistic notions. Primary screening was performed in a single-siRNA-per-well format with the human druggable genome siRNA set V4 comprising 13,984 siRNA targeting 6,992 genes and comprising two RNAi per gene. siRNA were transfected at low concentration (13nM) to minimize off-target effects using conditions that resulted in transfection of >95% cells and <5% background cytotoxicity. Bortezomib was added (at the IC10, 25, 70 and 90) 24 hours post transfection. After 96 hours (72 hours after bortezomib), viability was measured by ATP-dependent luminescence. In primary screening 320 candidate bortezomib sensitizing genes were identified and rescreened with four siRNA oligos each gene. 57 of the top sensitizer hits were selected for which at least two distinct siRNA decreased the EC50 by 2 standard deviations from cells treated with control siRNAs. By cross referencing gene expression profile (GEP) data of KMS11, we furthered pared the list to 37 plausibly expressed targets (0.5% of those genes originally screened) as bortezomib sensitizers. After silencing, 50% of these 37 genes also sensitized the lung cancer cell line A549 to bortezomib. After further parsing of genes which also modulated the sensitivity of MM to Melphalan (non specific chemosensitizers), 34 genes remained: The strongest sensitizers to bortezomib were the proteasome subunits PSMA5, PSMB2, PSMB3, PSMB7 but included less obvious targets such as BAZ1B, CDK5, CDC42SE2, MDM4, NME7, TFE3, TNFAIP3, TNK1, TOP1, VAMP2 and YY1 were also identified. Of these, the most potent synergetic effects were observed with siRNAs against the proteasome subunits and against cyclin dependent kinase 5 (CDK5), which caused the greatest shift in EC50. CDK5 is of particular interest as a therapeutic target as it is expressed at high levels in MM and neural tissues but has low expression in other organs. Using viral shRNA expression, silencing CDK5 consistently increased the sensitivity of genetically variable MM cell lines (n=5) to all of the proteasome inhibitors tested: bortezomib, carfilzomib and PR047, and the effect could be at least partially rescued by overexpression of an RNAi resistant CDK5. To explore therapeutic relevance the small molecule CDK5 inhibitor, Roscovitin, was shown to be synergistic or additive with bortezomib in both MM cell lines and primary patient samples. Gene expression profiling was then performed to seek an explanation for the CDK5 sensitization effect and regulation of a proteasome subunit PSMB5 by CDK5 was identified as a probable route to sensitization. In summary inhibition of the existing proteasome either directly by suppression of proteasome subunits, or indirectly by suppression of modulators such as CDK5 appears to confer the greatest sensitization effect suggesting that combinations of bortezomib with other unique proteasome inhibitor drugs or combinations with inhibitors of CDK5 is a logical avenue for clinical exploration. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 127 The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their anti-tumor effects remains unclear. A landmark paper has recently identified cereblon (CRBN) as a primary target of thalidomide teratogenicity. We hypothesized that this protein would also be required for myeloma (MM) cytotoxicity. To examine this issue we first tested 12 MM cell lines: 4 of which (OCIMY5, OPM1, SKMM2 and KMS12 PE) were very resistant to treatment with both lenalidomide and pomalidomide. Of these, OCIMY5 and OPM1 showed CRBN gene expression levels at the bottom 10% across the human MM cell lines. We next used CRBN shRNA lentiviral expression constructs to knock down CRBN in 5 human-derived myeloma cell lines (HMCLs) (KMS18, MM1.S, H929, OPM2 and JJN3). HMCLs infected with CRBN shRNAs have a significant reduction of CRBN expression and subsequent diminished cell viability (reduced 65–78%). Surviving MM cells with CRBN knock down demonstrate an acquired (essentially complete) resistance to lenalidomide when compared with controls but retained sensitivity to melphalan, dexamethasone and bortezomib. Gene expression changes induced by lenalidomide were dramatically suppressed in those CRBN depleted cells, e.g., OPM2 cells only showed 30 down regulated (3% of control) and 150 up-regulated genes (24% of control) after lenalidomide treatment, further emphasizing that CRBN is required for lenalidomide activity. We next compared by array comparative genomic hybridization the differences between MM1.S (lenalidomide sensitive) and its isogenic lenalidomide resistant line (MM1.S res), generated by culturing MM1.S in gradually increasing concentrations of lenalidomide. Only three structural abnormalities were noted differentiating the two lines one of which was deletion of CRBN. In fact the baseline MM1.S cell line has a minor population of 9% of cells with CRBN depletion which becomes dominant after lenalidomide treatment. However, further scrutiny of CRBN status in additional HMCLs and MM patients (irrespective of therapy and stage) suggested that copy-number abnormalities affecting this gene are uncommon events in MM, with 12% of HMCLs (7 out of 60) and only 1.2% of MM patients (3 out of 238) showing CRBN monoallelic deletion. Next, we examined by quantitative polymerase chain reaction (Q-PCR) ten MM patients who had become resistant to lenalidomide therapy. CRBN expression level by Q-PCR in 8 of 10 patient samples had a significant reduction (20% to 90% reduction compared to baseline) at time of drug resistance, suggesting CRBN expression as a potential biomarker to predict IMiD response. Notably however some clearly resistant patients have normal CRBN, demonstrating that while CRBN is absolutely required for response, multiple resistance mechanisms are likely present. By gene and protein expression downstream targets of CRBN were examined and included interferon regulatory factor 4 (IRF4) previously reported to also be a target of lenalidomide. In summary, we demonstrate here that CRBN is essential for IMiD activity and preliminary data support that low levels of CRBN predict for poor drug response. In addition, our data suggest that CRBN is a critical molecule but not the unique source of IMiD resistance. We suggest that the “IMiDs” be renamed Cereblon binding small molecules to more accurately reflect their mechanism of action. Disclosures: Orlowski: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.