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Introduction: Emerging evidence supports that cytogenetic abnormalities (CAs) drive myelomagenesis and heterogeneity (e.g., clinical presentation, response to therapy, and prognosis) of multiple myeloma (MM). Diversity of CAs, including primary (IgH translocations and trisomies) and secondary CAs (copy number abnormalities) argues that MM is not a "single disease". Thus, identification of CAs at diagnosis is essential for risk stratification, guiding treatment, and prognostic estimation in daily practice. However, although frequency, configuration, and significance of CAs have been well documented in the Western countries, this information is lacking in the Asian population. To this end, a multi-center retrospective analysis was carried out to examine epidemiology and prognostic significance of CAs alone or in combination in a cohort of Chinese patients with newly-diagnosed MM (NDMM). Materials and Methods: A total of 1015 NDMM patients who had the baseline information of CAs detected by FISH at four institutes nationwide were included. According to the IMWG consensus updated in 2016, 1q gain, del(17p), t[4;14], and t[14;16] were defined as high-risk CAs (HRCAs), while another HRCA t[14;20] was not tested routinely in a majority of these patients. In addition, del(1p) and del(13q14) were considered as an adverse CA. According to the mSMART3.0 proposed by Mayo Clinic in 2018, double-hit (DHMM) and triple-hit MM (THMM) were defined as co-occurrence of 2 or >= 3 HRCAs, respectively. The Kaplan-Meier approach was used to estimate progression-free survival (PFS) and overall survival (OS). Results: In this cohort, the median age of 1015 patients was 61 years; 63.5% were male. The type of IgG, light chain, IgA, IgD, non/oligosecretory, IgM, or IgE accounted for 42.9%, 26.2%, 24.5%, 3.4%, 2.5%, 0.4%, and 0.1%, respectively. Del(13q) (46.4%) and 1q gain (46.1%; 3 copies = 73.8%, >= 4 copies = 26.2%) represented the most common CAs, followed by t(4;14) (14.0%), t(11;14) (11.8%), del(1p) (11.5%), del(17p) (9.9%; 20-50% cells = 35.6%, > 50% cells = 64.4%), and t(14;16) (5.1%). While none of these CAs was detected in 23.8% of cases, the frequency of patients who carried 1 - 5 CAs was 31.9%, 28.0%, 13.4%, 2.0%, and 0.9%, respectively. In the 1q+ cases, 36.4% patients carried second CA(s), including del(13q) (61.1%), t(4;14) (20.3%), del(1p) (14.8%), del(17p) (10.7%), t(11;14) (10.4%), and t(14;16) (8.1%). In the del(17p) cases, 57.5% patients had additional CA(s), including del(13q) (75.2%), 1q gain (49.5%), del(1p) (21.6%), t(4;14) (19.6%), t(14;16) (8.7%), and t(11;14) (2.2%). In the cases bearing IgH translocations, 26.0% patients also carried other CA(s), including del(13q) and 1q gain (61.9% for each), and del(17p) (9.3%). In the cases harboring HRCAs, the percentage of patients who carried 1 - 4 HRCAs was 70.2%, 24.8%, 3.7%, and 1.1%, respectively. Overall, 14.3% and 2.9% patients had DHMM or THMM, of whom 65.0%, 18.0%, 12.0%, and 5.0% had 2 - 5 HRCAs, respectively. While there was no significant difference in PFS between the cases carrying 1 and 2 CAs (P = 0.209), the patients who had 3 or more CAs displayed a sharp reduction in median PFS (P = 0.022 and P = 0.003 for 3 vs 1 or 2 CAs). Although multiple CAs was associated with shorter median OS, no statistical significance was observed for each comparison (P > 0.05). However, patients who carried >= 2 HRCAs had significantly shorter median PFS (12.1 months; P = 0.0004) and OS (29.3 months; P = 0.027) than those who had one single HRCA (32.2 and 65.6 months for median PFS and OS). Conclusion: In comparison with the Western countries, the incidence of secondary HRCAs (e.g., 1q gain and del(17p)) is relatively higher in Chinese patients at diagnosis, while the standard-risk CA such as t(11;14) is clearly less frequent. The proportion of Chinese patients who carry multiple CAs (up to 5) or HRCAs appear to be greater as well. In this context, patients carrying two or more HRCAs, so called DHMM or THMM, exhibit significantly worse outcome than those carrying only one HRCA. Together, this study builds up an up-to-date profile of CAs for Chinese patients, which might lay a foundation for revising the criteria for risk stratification and the guideline for treatment that is more feasible and practicable in China. It also provides the information about frequency and configuration of MM-driven CAs, which might be more relevant to the Asian population. Disclosures Kumar: Takeda: Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding.

Abstract Pathogenic sequence variants (PSV) in BRCA1 or BRCA2 (BRCA1/2) are associated with increased risk and severity of prostate cancer. We evaluated whether PSVs in BRCA1/2 were associated with risk of overall prostate cancer or high grade (Gleason 8+) prostate cancer using an international sample of 65 BRCA1 and 171 BRCA2 male PSV carriers with prostate cancer, and 3,388 BRCA1 and 2,880 BRCA2 male PSV carriers without prostate cancer. PSVs in the 3′ region of BRCA2 (c.7914+) were significantly associated with elevated risk of prostate cancer compared with reference bin c.1001-c.7913 [HR = 1.78; 95% confidence interval (CI), 1.25–2.52; P = 0.001], as well as elevated risk of Gleason 8+ prostate cancer (HR = 3.11; 95% CI, 1.63–5.95; P = 0.001). c.756-c.1000 was also associated with elevated prostate cancer risk (HR = 2.83; 95% CI, 1.71–4.68; P = 0.00004) and elevated risk of Gleason 8+ prostate cancer (HR = 4.95; 95% CI, 2.12–11.54; P = 0.0002). No genotype–phenotype associations were detected for PSVs in BRCA1. These results demonstrate that specific BRCA2 PSVs may be associated with elevated risk of developing aggressive prostate cancer. Significance: Aggressive prostate cancer risk in BRCA2 mutation carriers may vary according to the specific BRCA2 mutation inherited by the at-risk individual.

Abstract Background Infection occurs in ~1–2% of prosthetic joint replacement surgeries, with staphylococci being the most common cause. TNP-2092 is an investigational drug composed of rifamycin and quinolizinone pharmacophores conjugated via a stable linker. Here, we determined TNP-2092’s in vitro activity against biofilms formed by staphylococci associated with prosthetic joint infection and compared activity to that of ciprofloxacin and rifampin alone and in combination, as well as to daptomycin and vancomycin. Methods A total of 80 staphylococcal isolates (40 Staphylococcus aureus and 40 Staphylococcus epidermidis) were studied. Planktonic state minimum inhibitory concentrations (MICs) of TNP-2092, ciprofloxacin, rifampin, ciprofloxacin + fixed concentration (1 mg/mL) rifampin, daptomycin and vancomycin were determined following CLSI guidelines. Tween-80（0.002%）was added to TNP-2092 to prevent drug binding to plastic plates. Minimum biofilm inhibitory concentrations (MBICs) and minimum biofilm bactericidal concentration (MBBCs) were determined as follows. Bacteria were grown in TSB to logarithmic phase and adjusted to a turbidity of 0.5 McFarland; 150 µL aliquots were transferred to individual wells of 96-well flat-bottom plates and the plates covered with 96-pegged lids. Plates were incubated on a shaker for 5 hours at 37℃. Pegged lids were rinsed using 200 µL PBS/well and placed into a microtiter plate containing serial 2-fold drug dilutions in CAMHB Plates were incubated for 20–24 hours at 37°C and MBICs read by visual turbidity. Pegged lids were rinsed with PBS and placed into plates filled with 200 µL CAMHB/well and incubated for 20–24 hours at 37°C after which MBBCs were determined by assessing visual turbidity. Results Results shown in the table. Conclusion TNP-2092 has promising in vitro activity against prosthetic joint infection-associated staphylococcal biofilms. Disclosures Robin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.

AbstractNF-κB and Notch signaling can be simultaneously activated in a variety of B-cell lymphomas. Patients with B-cell lymphoma occasionally develop clonally related myeloid tumors with poor prognosis. Whether concurrent activation of both pathways is sufficient to induce B-cell transformation and whether the signaling initiates B-myeloid conversion in a pathological context are largely unknown. Here, we provide genetic evidence that concurrent activation of NF-κB and Notch signaling in committed B cells is sufficient to induce B-cell lymphomatous transformation and primes common progenitor cells to convert to myeloid lineage through dedifferentiation, not transdifferentiation. Intriguingly, the converted myeloid cells can further transform, albeit at low frequency, into myeloid leukemia. Mechanistically, coactivation of NF-κB and Notch signaling endows committed B cells with the ability to self renew. Downregulation of BACH2, a lymphoma and myeloid gene suppressor, but not upregulation of CEBPα and/or downregulation of B-cell transcription factors, is an early event in both B-cell transformation and myeloid conversion. Interestingly, a DNA hypomethylating drug not only effectively eliminated the converted myeloid leukemia cells, but also restored the expression of green fluorescent protein, which had been lost in converted myeloid leukemia cells. Collectively, our results suggest that targeting NF-κB and Notch signaling will not only improve lymphoma treatment, but also prevent the lymphoma-to-myeloid tumor conversion. Importantly, DNA hypomethylating drugs might efficiently treat these converted myeloid neoplasms.

Abstract Introduction: Mutations localized in the tyrosine kinase domain activation loop of FLT3 (FLT3-TKD), representing point mutations in codon D835/I836 and rarely deletions of codon I836, induce constitutive tyrosine phosphorylation and activation of the receptor tyrosine kinase similarly to FLT3 internal tandem duplication (ITD) mutations. However, the prognostic role of FLT3-TKD in AML, particularly in the presence of NPM1 mutations, is not well established. The phase 3 RATIFY trial [NCT00651261; Stone et al. N Engl J Med. 2017] showed that in combination with standard chemotherapy, midostaurin (PKC412) improved survival outcomes across all 3 FLT3 stratification subgroups (ITD high allelic ratio [≥ 0.7], ITD low allelic ratio [< 0.7], and TKD) vs placebo in patients with newly diagnosed FLT3-mutated AML. Here, we evaluated the prognostic impact of FLT3-TKD and NPM1 mutations in a post hoc analysis from the RATIFY trial. Methods: In RATIFY, newly diagnosed patients with AML 18-60 years old were randomly assigned to receive midostaurin or placebo together with standard induction and consolidation therapy followed by 12 28-day cycles of maintenance therapy with midostaurin or placebo. FLT3-TKD mutation was detected by PCR and capillary electrophoresis at 9 reference laboratories. Patients were categorized as NPM1 mutated (mut) or NPM1 wild-type (WT) using PCR. Efficacy outcomes included complete remission (CR), overall survival (OS), event-free survival (EFS) and disease-free survival (DFS). EFS and DFS analyses were performed considering CR within a 60-day window. P values presented have not been adjusted for multiplicity. Results: Of the total randomized 162 FLT3-TKD patients, 134 with available NPM1 data had consented for exploratory analysis and thus were included in this study (see Table for subgroup distribution). Overall, 47.8% of patients were male, and the median age was 49 years (95% CI, 45.5-51.1 years). The median white blood cell (WBC) count was higher in patients with NPM1-mut than in patients with NPM1-WT (34.1 vs 15.5 × 109/L, P = .0011). CR rates (during the first 60 days) were higher in patients with FLT3-TKD/NPM1-mut vs FLT3-TKD/NPM1-WT (66% vs 53%); however, this was driven by the higher rate of CR in the midostaurin arm (76% NPM1-mut vs 44% NPM1-WT) rather than the placebo arm (53% NPM1-mut vs 60% NPM1-WT). The overall CR rate (regardless of NPM1 genotype) was 64% for midostaurin and 56% for placebo in FLT3-TKD patients. The prognostic effect of the NPM1 mutation concurrent with FLT3-TKD was seen for all endpoints consistently with hazard ratios (HRs) around 0.50 or lower (Figures 1 and 2 and Table). Overall (regardless of treatment) OS, EFS, and DFS estimates at 3 years were 73% vs 52%, 48% vs 25%, and 74% vs 47%, respectively, in patients with FLT3-TKD/NPM1-mut vs FLT3-TKD/NPM1-WT. Whereas the HRs for midostaurin vs placebo were 0.73 for both OS and EFS, the impact of treatment on outcomes varied between the individual NPM1/TKD subgroups and was not consistently observed when endpoints were censored at stem cell transplant (SCT) (Table). It should be noted that the number of patients in each subgroup was small and therefore the HRs with 95% CIs should be interpreted with caution. Multivariate analyses in these FLT3-TKD patients revealed that NPM1 genotype was an independent prognostic factor for OS, EFS and DFS (2-sided P < .05), whereas study drug, age, sex, WBC at baseline and SCT (no/yes) did not reach this level of significance in the Cox model. Conclusions: This post hoc analysis of the FLT3-TKD patient subset in the RATIFY trial showed the high prognostic value of NPM1 mutational status. Whereas midostaurin showed an overall benefit in the FLT3-TKD patients for OS, EFS, CR and DFS, the impact of treatment on outcome varied between the individual NPM1 subgroups within these FLT3-TKD patients and was not consistently observed.Further analyses using additional endpoints and additional multivariate analyses are planned. Support: U10CA180821, U10CA180882, U10CA180820, U10CA180791, U10CA180888, U10CA180863, (CCSRI) #704970, U24CA196171; ClinicalTrials.gov Identifier: NCT00651261 Disclosures Voso: Celgene: Research Funding, Speakers Bureau. Larson:Ariad/Takeda: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BristolMyers Squibb: Consultancy, Research Funding. Heuser:Janssen: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; StemLine Therapeutics: Consultancy; Astellas: Research Funding; BergenBio: Research Funding; Karyopharm: Research Funding; Bayer Pharma AG: Consultancy, Research Funding; Tetralogic: Research Funding; Sunesis: Research Funding; Daiichi Sankyo: Research Funding. Wei:Novartis: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau; Pfizer: Honoraria, Other: Advisory committee; Amgen: Honoraria, Other: Advisory committee, Research Funding; Abbvie: Honoraria, Other: Advisory board, Research Funding, Speakers Bureau; Servier: Consultancy, Honoraria, Other: Advisory committee, Research Funding; Celgene: Honoraria, Other: Advisory committee, Research Funding. Brandwein:Lundbeck: Consultancy; Celgene: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Boehringer Ingelheim: Consultancy, Research Funding. de Witte:Novartis: Research Funding; Amgen: Consultancy, Research Funding; Celgene: Honoraria, Research Funding. Medeiros:Celgene: Consultancy, Research Funding; Genentech: Employment. Tallman:Cellerant: Research Funding; Orsenix: Other: Advisory board; BioSight: Other: Advisory board; ADC Therapeutics: Research Funding; AROG: Research Funding; AbbVie: Research Funding; Daiichi-Sankyo: Other: Advisory board. Schlenk:Pfizer: Research Funding, Speakers Bureau. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Cheng:Novartis: Employment. Gathmann:Novartis: Employment. Tiecke:Novartis: Employment. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding. Döhner:AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Celator: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Agios: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Pfizer: Research Funding; Pfizer: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Stone:Otsuka: Consultancy; Argenx: Other: Data and Safety Monitoring Board; Amgen: Consultancy; Agios: Consultancy, Research Funding; Orsenix: Consultancy; Ono: Consultancy; Novartis: Consultancy, Research Funding; Astellas: Consultancy; Arog: Consultancy, Research Funding; Merck: Consultancy; Cornerstone: Consultancy; Fujifilm: Consultancy; Jazz: Consultancy; Celgene: Consultancy, Other: Data and Safety Monitoring Board, Steering Committee; Pfizer: Consultancy; Sumitomo: Consultancy; AbbVie: Consultancy.

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.