Stewart, Keith

Abstract In an attempt to identify novel genetic abnormalities in multiple myeloma (MM) we analyzed 68 MM patient samples and 42 human myeloma cell lines (HMCL) with high-resolution (60kb) array-based comparative genomic hybridization (aCGH). We concentrated on bi-allelic genomic deletions with the expectation that these regions might contain novel tumor suppressor genes and identified a common abnormality at 14q32. Fine mapping of the 14q32 microdeletion by PCR demonstrated a 45 kb minimal deletion encompassing two genes; TRAF3, a negative regulator of the non-canonical NF-kB pathway, and AMN. To determine the frequency of copy number abnormalities of this region a cohort of 161 patients were screened with a FISH probe mapping to the minimally deleted region. This identified 7 (4.4%) patients with bi-allelic deletion and 19 (11.8%) patients with a single copy of the target region. Sequencing of all TRAF3 coding exons in 62 patients and 42 HMCL identified inactivating mutations in 9 patient samples and 4 HMCL. Overall, the incidence of TRAF3 inactivation, bi-allelic deletion or mutation, is 7/42 (16.7%) in HMCL and 12/62 (19.4%) in patients, making it one of the most common genetic abnormalities identified in MM. Importantly, dysregulation of NF-kB pathways is a common event in MM, but the mechanism for this dysregulation is largely unknown. Although TRAF3 is a negative regulator of the non-canonical NF-kB pathway it has not previously been implicated in cancer. Therefore, we tested the tumor suppressor function of TRAF3 by reintroducing TRAF3 into HMCL harboring TRAF3 abnormalities using an adenovirus gene transfer system. Expression of TRAF3 in HMCL with TRAF3 abnormalities rapidly reversed cellular processing of NFKB2 from p100 (an inactive isoform) to p52 (active) and additionally led to a 60% reduction in cellular proliferation (range, 10–89%) associated with Go/G1 cell cycle arrest and induction of cell death by apoptosis. At 48 hrs post infection the proportion of cells entering S-phase or G2/M declined by 31% (range, 8.1–56.1) and the proportion undergoing apoptosis increased by 28% (range 11.7–47.4%). In contrast, over-expression of TRAF3 in control HMCL without TRAF3 abnormalities had no effect. Overall, TRAF3 is identified as a novel tumor suppressor that regulates the non-canonical NF-kB pathway and is inactivated in ~ 20% of MM patients. Given the involvement of the NF-kB pathway in multiple human malignancies and the near constitutive expression of TRAF3 we propose that inactivation of TRAF3, a novel tumor suppressor identified for the first time in this study, may occur in other cancers. In this regard, we have recently identified a Burkitt lymphoma cell line that does not express TRAF3 and thus has constitutive processing of p100 to p52. The clinical and therapeutic consequences of this finding and its frequency in other tumor types are currently the focus of further investigation. In conclusion, the abnormalities identified in this study represent the first mutations identified that cause constitutive NF-kB activation in MM.

Abstract The t[4;14] simultaneously deregulates fibroblast growth factor receptor 3 (FGFR3) and MMSET in 15% of patients (pts) with Multiple Myeloma (MM). Although oncogenic, FGFR3 expression is lost in ~25% of t[4;14] pts on gene expression profiling while MMSET is always retained. The t[4;14] is highly associated with poor prognosis, furthermore FGFR3 kinase inhibitors are in Phase I trials, thus detection of t[4;14] pts is increasingly important. The Multiple Myeloma Research Consortium (MMRC) therefore evaluated 4 competing methodologies for the diagnostic detection of t[4;14]. Bone marrow (BM) samples were collected in uniform fashion from 85 pts. FGFR3 immunocytochemistry (ICC) and flow cytometry (FC) were performed at the retrieval site, while split samples were shipped to the MMRC tissue bank and processed under GLP conditions. Processed samples were analyzed by cIg-FISH and quantitative IgH-MMSET RT-PCR (QPCR) on unsorted BM and blood. Of 60 samples with sufficient plasma cells for analysis, 8 were FISH +ve [13.3%]. Low % BM plasma cells prevented successful FISH in 25% of samples reflecting large volume harvests and hemodilution. Analysis was conducted in a blinded fashion. With FISH as the gold standard for detection of t[4;14] the sensitivity and specificity of the other diagnostic tests are: BM QPCR is most sensitive (7 of 8 FISH positive detected) and specific (42/43 negatives correctly identified). QPCR on peripheral blood is ongoing. FC is more sensitive to the detection of FGFR3 protein than ICC. Correlation between FC and ICC was only 0.46. Interestingly, 6 of 7 evaluable pts here and 13/14 (92%) in an expanded analysis of FISH +ve patients also expressed the FGFR3 protein. Given the discrepancy between this finding and previously reported loss of FGFR3 expression in 25% of pts we further explored t[4;14] stability. Clonal selection/heterogeneity was determined by the % plasma cells in each pt. with an unbalanced translocation (loss of one der chromosome). In 42 t[4;14] pts, 13 (31%) had a balanced translocation(>75% of cells with a double fusion), 14 (33%) had an unbalanced translocation (>75% cells with only one signal), and 15 (36%) had a chimeric picture. This heterogeneous pattern is suggestive of an evolution towards an unbalanced translocation. Despite this survival did not differ between pts with balanced or unbalanced translocation. Conclusions: These results indicate that the t[4;14] is highly heterogeneous with respect to balanced versus unbalanced translocations, which likely evolve over time. For detection, cIg-FISH continues to be the gold standard diagnostic methodology but may be limited by low plasma cell numbers in dilute BM or during MM remission. QPCR and FC appear most sensitive and specific for presence of t[4;14] and FGFR3 protein expression respectively. Surprisingly (given prior evidence of loss of FGFR3 expression in 25% of pts) 92% of t[4;14] pts in this series expressed FGFR3 protein. # Analyzed % Positive Sensitivity Specificity FISH 60 13 - - QPCR 65 14 87.5 97.6 Flow 82 15 85.7 91 ICC 85 15 62.5 92

Abstract Abstract 1807 We have recently demonstrated that cereblon (CRBN) mediates the direct anti-myeloma activity of immunomodulatory drugs (IMiDs). However, the genes/pathways downstream of CRBN associated with anti-myeloma activity remain unclear. We, and others, identified interferon regulatory factor 4 (IRF4) as one of the downstream targets of CRBN-associated signaling. Both lenalidomide treatment and CRBN knockdown downregulate IRF4. IRF4 levels return to baseline in IMiD resistant cells surviving CRBN silencing. To determine whether IMiD-induced IRF4 downregulation is critical to anti-MM activity, we overexpressed IRF4 in two IMiD-sensitive human MM cell lines (HMCLs), KMS11 and MM1.S, followed by lenalidomide treatment. Lenalidomide-induced cytotoxicity was greatly impaired in both HMCLs overexpressing IRF4 compared with the control virus infected cells. Further analysis indicated that IRF4 over-expression does not completely prevent lenalidomide-induced growth arrest, but reduces cell death by 70% after lenalidomide treatment. Immunoblotting analysis of KMS11 cells indicated that IRF4 over-expression blocks lenalidomide-induced activation of caspase 8, reduces up-regulation of p21waf and increases CDK6 expression but does not significantly affect lenalidomide-induced MYC down-regulation. Although cereblon and IRF4 are broadly expressed in MM, baseline levels of expression are only weakly correlated (r=0.22) in primary MM patient gene expression analysis. Gene expression studies revealed statistical changes in 1,368 genes when comparing high versus low CRBN expression in primary myeloma samples. Interestingly genes associated with high CRBN expression included cyclin D2, SOCS3 and IL4 while genes associated with low cereblon expression included cyclin D1, FRZB and CD200. In order to understand how CRBN is connected with downstream anti-myeloma signaling, a structure-function study was performed to determine which CRBN domain is required for lenalidomide-induced IRF4 down-regulation and cytotoxicity. Lentiviral constructs expressing wild-type CRBN and a series of mutated CRBN were generated, including mutations at thalidomide binding site (Y384A/W386A), deletion of DDB1 binding region (ΔMid) and truncations at N-terminal and C-terminal. Lentiviruses from these constructs were used to infect IMiDs resistant HMCLs, OCI-MY5 and MM1.S res. Both of these cell lines have very low endogenous CRBN expression and they became sensitive to lenalidomide after introduction of wild-type CRBN. Conversely, introduction of CRBN with mutated thalidomide-binding site or with DDB1 binding region depletion failed to mediate lenalidomide toxicity and down-regulation of IRF4. OCI-MY5 cells expressing either N-terminal or C-terminal truncated CRBN showed substantial reduced responses (more than 50%) to lenalidomide compared with wild-type CRBN expressing cells. Deletion of only 20–30 amino acids at either ends of CRBN greatly impaired the protein function, suggesting that protein folding might be important for CRBN-mediated IMiD response. Our data indicate that IMiD induced myeloma cytotoxicity is largely mediated by modifying CRBN associated E3 ubiqutin ligase and subsequent IRF4 downregulation, suggesting the CRBN-IRF4 axis is a potential target for development of new anti-myeloma drugs. Disclosures: Schmidt: Karyopharm: Research Funding. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy.

Abstract Abstract 1793 Thalidomide (THAL) and IMID® immunomodulary drugs lenalidomide (LEN) and (POM) have proven beneficial in the treatment of a variety of hematological malignancies. Pre-clinical studies demonstrate multiple direct and indirect anti-tumor activities including anti-angiogeneic, proapoptotic, anti-proliferative and immunomodulatory effects. Recent studies have identified cerebron (CRBN) as a potential direct physical target of THAL and IMiD compounds and CRBN expression is reportedly required for IMiD compound activity. However, the precise link between CRBN and IMiD compound mechanism of action (MOA) have not been clearly defined. We applied Drosophila as a drug discovery platform to assess the MOA of THAL and IMiD compounds in vivo. THAL or POM fed Drosophila demonstrate morphological phenotypes that replicate wingless (wg) mutants indicating that drugs are inhibitors of Wg/Wnt signaling. In this model system, THAL and IMiD compounds disrupt membrane localization of GSK-3 indicating that the bioactivity of IMiD compounds is achieved through spatial regulation and potentiation of Sgg/GSK-3 in Drosophila. Using epistasis analysis we show that Drosophila expressing genetic mutants lacking GSK-3 activity and myeloma cells in which GSK-3a and GSK-3b have been knocked down by siRNA fail to respond to POM. In both Drosophila and myeloma cells therefore it appears that GSK-3 activity is required for biological responses. To test the clinical validity of GSK-3 as a biomarker, we obtained patient tumor samples from a Phase II clinical trial of single agent LEN for previously untreated CLL (Chen CI et al., J Clin Oncol, 2011; 29:1175). Twenty five patients were enrolled onto this study and received LEN at a starting dose of 2.5 mg days 1–21 of a 28 day cycle with monthly escalation to a target dose of 10 mg. The primary clinical endpoint for the trial was objective response to lenalidomide (complete response (CR) and partial response (PR)) evaluated as defined in the revised 1996 NCI Working Group guidelines. Peripheral blood samples for correlative studies were collected on days 1 (pre-dosing) and 8 of cycles 1 and 2. CRBN expression was evaluated by gene expression profiling and Western blot and found to be uniformly expressed in all 19 evaluable day 1 patient samples regardless of LEN response. Thus CRBN expression does not appear to be a useful predictive biomarker of response in this population of previously untreated patients. However, GSK-3 localization was correlated with response. Paired analysis of CD19 selected CLL cells comparing day 1 vs day 8 revealed focal membrane localization of GSK-3 on day 1 and subcellular redistribution on day 8 in 11 out of 12 evaluable responders (PR or better). By contrast, in the CLL cells from all 6 evaluable non-responders, GSK-3 expression appeared diffusely distributed before and after treatment. In preliminary studies using confocal immunofluorescence microscopy we determined that CRBN and GSK-3 co-localize in day 1 CLL samples of responders but not in those of non-responders. In summary, our results indicate that THAL and the IMiD compounds target GSK-3 function by spatial regulation and identify GSK-3 localization as a potential clinical biomarker of IMiD response. Disclosures: Trudel: Celgene: Honoraria; GlaxoSmithKline: Research Funding; Janssen: Honoraria. Mercurio:Celgene: Equity Ownership, Research Funding. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Gaidarova:Celgene Corp: Employment, Equity Ownership. Webb:Celgene: Employment, Equity Ownership. Chen:Johnson & Johnson: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Lundbeck: Consultancy. Stewart:Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy. Manoukian:Celgene: Research Funding.

Abstract Introduction A unique biological characteristic of MM plasma cells (PCs) is the substantial production of immunoglobulin proteins. This process requires disulfide bond formations that in turn generate large amounts of H2O2. The Trx-TrxR reductase system exists to combat this oxidative stress; however, cytotoxicity can occur when the cells' antioxidative capacity is overwhelmed. Recently, we demonstrated that IMiDs inhibit peroxidase-mediated decomposition of H2O2, and that lenalidomide sensitivity in MM correlates with the degree of antioxidative capacity (Sebastian et al. 2015 ASH). In this study we sought to better understand this inhibitory process. Methods We used various human MM cell lines with different sensitivity to lenalidomide. Cell viability was assessed by the MTT assay. Antioxidative capacity was measured via the total cellular oxidation of reduced flavin adenine dinucleotide (FADH2) and reduced nicotinamide adenine dinucleotide (NAD(P)H) after H2O2treatment. A CRBN CRISPR/Cas9 KO plasmid was used to generate CRBN knockout cells and lentivirus system was used to over-express CRBN in MM cells. Results We found that IMiDs inhibit TrxR mediated intracellular H₂O₂ decomposition in a CRBN dependent fashion. Unlike lenalidomide, the direct TrxR inhibitor auranofin was very effective in inducing cell death in MM cell lines, in a CRBN independent fashion. By using MM.1R isogeneic cells with and without CRBN, and the CRBN-overexpressing OCIMY-5 cell line (transfected with wt-CRBN) we showed that auranofin treatment caused cytotoxicity irrespective of CRBN level of expression, while lenalidomide was only effective in CRBN expressing cells. Clinically achievable concentrations of auranofin treatment induced cell signals similar to lenalidomide, including degradation of IKZF1, IKZF3. As with lenalidomide, the sensitivity of CRBN expressing cell lines to auranofin correlated with their basal antioxidative capacity (MM.1S cells are sensitive and have with lower anti-oxidative capacity while RPMI-8226 is resistant due to its high anti-oxidative capacity). We also found that targeting Trx (downstream effector of TrxR) with the direct inhibitor PX12 overcame this cellular anti-oxidative capacity and inhibited cell proliferation in both RPMI-8226 and MM.1S in a CRBN independent fashion. Combination of auranofin with bortezomib showed a synergistic effect, but only in cells with lower anti-oxidative capacity. In contrast, PX-12, showed a synergistic effect with bortezomib in all analyzed MM cells lines, irrespective of their anti-oxidative capacity and CRBN expression. Conclusions IMiDs inhibit TrxR-mediated intracellular H2O2decomposition in CRBN expressing MM. While the direct TrxR inhibitor, auranofin, bypassed the CRBN requirement for inducing cytotoxicity in MM, its cytotoxic effect could be overcome by the cells antioxidative capacity. Direct Trx inhibition (such as PX-12) seems like a potent strategy for MM therapy to overcome cellular CRBN requirements and higher anti-oxidative capacity cells. Disclosures Bergsagel: Novartis: Research Funding; Amgen, BMS, Novartis, Incyte: Consultancy. Stewart:celgene: Consultancy. Fonseca:Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Janssen: Consultancy; Bayer: Consultancy; Millennium, a Takeda Company: Consultancy; Millennium, a Takeda Company: Consultancy; BMS: Consultancy; Millennium, a Takeda Company: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Celgene: Consultancy; AMGEN: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Millennium, a Takeda Company: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Janssen: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Bayer: Consultancy; AMGEN: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Novartis: Consultancy; AMGEN: Consultancy; Sanofi: Consultancy; AMGEN: Consultancy; Sanofi: Consultancy; BMS: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms.

Abstract Abstract 1846 Background The low frequency of TP53 (p53) alterations (<10%) in MM makes this tumor an ideal candidate for p53-activation therapies. Unfortunately, DNA alkylating agents (cyclophosphamide and melphalan), when used alone, cannot fully exploit p53 mediated apoptosis. It is currently unknown the extent to which DNA alkylating agents and p53 activating small molecules are activating functional p53 signaling to induce apoptosis in primary MM cells, because, p53 mainly induces cell cycle arrest/DNA repair or apoptosis in the DNA damage response. Emerging studies demonstrate that PARP activation and p53 poly(ADP-ribosyl)ation can negatively influence apoptosis induction but the exact molecular mechanisms are unknown. Methods For apoptosis and gene expression analysis 05 × 106 cells were incubated with 2.5 μM of melphalan, 10 μM of olaparib and 2.5 μM of nutlin-3a alone and/or combinations of melphalan with olparib and/or nutlin3a with olaprib for 24 to 72 hours before they were examined for cell death by the annexin-PI and FACS analysis. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. For combination studies, cells were incubated with 0.194–50 μM of melphalan and 0.625–40 μM of either olaparib or ABT-888 for 72 hours. p53 knock-down performed by lentiviral mediated shRNA. Vk*MYC mice with significant gammopathy (>20g/l on SPEP) used for in-vivo drug combination studies. Results Preliminary results support our principle hypothesis that, a PARP inhibitor (olaparib) enhanced Melphalan sensitivity in wild type p53 MM cell lines H929 and MM1S but not in p53 mutated or homozygously deleted cell lines U266 and KMS11, respectively. Moreover, we demonstrate that p53 knock-down decreased the synergistic effect of combining a PARP inhibitor with melphalan and Nutlin-3a. Again, combining a PARP inhibitor with the DNA alkylating agent melphalan induced clear PARP cleavage, a signature of apoptosis in wild type p53 MM cell lines H929 and MM1S. No significant PARP cleavage was observed after melphalan or olaparib treatment alone in H929 and MMIS. As expected, the p53 mutant cell line U266 did not show any PARP cleavage and p21 up-regulation after both drug treatments. Here we propose that the hyper-activated p53 from PARP inhibition along with DNA alkylating agents (melphalan) and/or p53 activating agents (nutlin-3a) result in better responses. Finally, in a murine xenograft model of human MM, olaparib potentiated melphalan activity in vivo, with significant reduction in M spike level. Conclusion Our studies indicate that DNA alkylating agents activate wild type p53, but leads to DNA repair and cell survival and this can be abolished by PARP inhibition in wild type p53 expressing MM cases. Moreover, our study also found that olaparib, and ABT-888 - PARP1 inhibitors currently in clinical trials - display different dose responses in MM cell lines and in the abilities of individual PARP inhibitors to sensitize MM cell lines to melphalan varied to a great extent in a cell context- and cell line specific manner. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2741 Background: The novel agent lenalidomide (LEN) has shown significant single-agent activity in patients with relapsed lymphoma through anti-proliferative and immunomodulatory mechanisms. In combination LEN may synergistically improve the efficacy of rituximab. However, there is limited data on the efficacy and safety of LEN in multi-drug combination regimens for treatment-naïve patients. We designed a phase II single arm trial to evaluate tumor response, toxicity (AE) and survival with a combination of LEN, rituximab, cyclophosphamide (CTX) and dexamethasone (DEX) (LR-CD) in symptomatic untreated patients with indolent B-cell lymphoma. (NCT00784927). Methods: Eligibility required age ≥18, ECOG PS ≤2, confirmed diagnosis of indolent B-cell lymphoma (FL1–2, SLL, MZL or LPL/WM), measurable nodes ≥2cm or IgM ≥400mg/dL (LPL/WM), ANC ≥1400/mm3, platelets ≥100,000/mm3, creatinine ≤2mg/dL, and signed informed consent. Only patients needing therapy were considered eligible. Treatment consisted of IV rituximab 375mg/m2 D1, oral LEN 20mg D1–21, CTX 250mg/m2 D1, 8, 15, DEX 40mg D1, 8, 15, 22 and ASA 325 mg daily in a 28 day cycle. Treatment continued 2 cycles beyond best response up to a maximum of 12 cycles. Toxicity was assessed by NCI CTCAE v3.0. Results: Thirty-one patients have enrolled at Mayo Clinic with 28 evaluable for toxicity and response (1 deemed ineligible, 1 withdrew before treatment, 1 treatment violation). Four patients continue on study at this time. Patient characteristics: median age 68.5(43–83), 71% male, stage IV 86%, FL 28%, MZL 25%, LPL/WM 39%, and SLL 4%. Median number of cycles given was 6 and 71% completed the study per protocol. The best overall response is 89% with 32% CR and 57% PR. In subgroup analysis, the response rates for LPL/WM was 91% (all PR) and for FL 88% (CR 63%, PR 25%). The most common grade ≥3 AEs were neutropenia (35.7%), leukopenia (17.8%), anemia (14.3%) and thrombosis (14.3%). The LPL/WM subgroup experienced more grade ≥3 anemia (36%) compared to the group as a whole. Patients (57%) required at least one dose reduction of LEN (n =10), CTX (n = 9) or DEX (n = 7). At a median f/u of 14.5 (1.8–36.5) months 86% are progression free (fig. 1) and the OS is 96% (fig. 2). One death unrelated to treatment occurred 7.7 months after completing 12 cycles of treatment. Conclusion: Lenalidomide can be safely combined with other active agents and in the combination LR-CD produces high response rates in symptomatic patients with indolent B-cell lymphoma. The regimen is well tolerated with manageable toxicities which are similar to that seen with single agent LEN. The encouraging results seen in patients with LPL/WM have led to expansion of this cohort for additional study. Disclosures: Reeder: Celgene: Research Funding. Off Label Use: lenalidomide use in non-Hodgkin lymphoma. Stewart:Celgene: Consultancy, Honoraria.

Abstract Multiple myeloma tumors universally target one of the three human cyclin D genes (CCND1, CCND2 or CCND3) for dysregulation (Bergsagel et al., 2005, Blood;106:296). To identify novel pharmaceutical inhibitors of cyclin D2 (CCND2) transactivation we therefore screened the Lopac, Prestwick and Spectrum libraries of drugs and natural compounds (n>4000) using NIH 3T3 cells stably expressing the CCND2 promoter driving a luciferase reporter gene. From this library screen and subsequent validation experiments we identified Kinetin riboside, a nucleoside analogue and plant cytokinin hormone, as a novel inhibitor of CCND1+CCND2 transactivation. By immunoblotting, Kinetin riboside induced rapid suppression of cyclin D1 and D2 proteins (<6 hours) in H929, JJN3, Kms11 and U266 human myeloma cell lines (HMCL) that all over-express either cyclin D1 or D2 due to a range of transforming events relevant to myeloma (including deregulation of FGFR3, MMSET or c-Maf oncogenes, or translocation of CCND1 to the IgH locus). Similar results were obtained in primary CD138+ purified myeloma cells from 5/6 patients. To verify that cyclin D1 and D2 suppression induced by Kinetin riboside is a direct effect and does not occur secondary to cellular arrest in S-phase (when cyclin D protein levels decline) we examined the effects of Kinetin riboside on the cell cycle profile of HMCL. Within 20 hours Kinetin riboside caused the proportion of cells entering S-phase to fall by 50–70% in all HMCL tested, consistent with primary cyclin D suppression and secondary G0/G1 arrest. By MTT assay, Kinetin riboside is cytotoxic to HMCL with an IC50 of <1.7 mg/L (5uM) in 8/12 lines and <5mg/L (15uM) in 11/12 HMCL. By comparison, toxicity studies in Balb/c mice confirm that Kinetin riboside is tolerated in vivo at a dose of 80–100mg/kg i.p. or 25mg/kg i.v. Importantly, Kinetin riboside shows potent synergy with dexamethasone in HMCL that are poorly responsive to one or other agent and Kinetin riboside activity persists during co-culture with myeloma growth factors IL-6, IGF-1 and Baff. Moreover, when tested against unsorted patient bone marrow samples, Kinetin riboside preferentially killed CD138+ myeloma cells at up to 5–8 fold greater rate than normal marrow progenitors. Kinetin riboside induced myeloma cell death is mediated by apoptosis and is associated with caspase 9 cleavage and annexin V binding. Mechanistically, we show that kinetin riboside blocks CCND2 promoter transactivation induced by cAMP or by regulatory phosphoproteins (activated by Forskolin or the PP2A inhibitor, Cantharadin, respectively) and also blocks cis-activation of CCND1 induced by translocation to the IgH enhancer and trans-activation of CCND2 induced by cMaf or FGFR3 over-expression, indicating that kinetin riboside acts at a distal level to block transactivation of CCND1+CCND2 induced by multiple factors. Gene expression profiling reveals that KinR causes rapid induction of the transcriptional repressor, cAMP Response Element Modifier (CREM), which has been reported to bind the cyclin D2 promoter to regulate cyclin D2 expression (Muniz, 2006, Biol Reprod.), providing a putative mechanism for targeted suppression of CCND1+CCND2. Together these studies demonstrate a novel targeted mechanism and substantial pre-clinical activity for kinetin riboside and provide a rationale for clinical evaluation of this drug to improve the outcome of multiple myeloma.

Abstract Next-generation sequencing (NGS) has revealed new insight into the complexity of clonal and subclonal architecture of multiple myeloma (MM). The introduction of targeted studies allows the detection of mutations with very low allele frequencies at affordable price. However, there is few information about the prognostic impact of this mutational profile in series of homogeneously treated MM patients. In this study, we analyzed the most frequently mutated genes in MM from patients included in a randomized clinical trial applying the highest read depth to date. We performed a correlation study between our results and cytogenetics aberrations and clinical outcomes. We used Ampliseq Library Kit 2.0 to target 77 genes (M3Pv2.0 panel) related to critical pathophysiological pathways, associated to drug resistance or targetable with molecular drugs in MM. We sequenced DNA (15ng) from CD138+ purified plasma cells from 80 MM patients at diagnosis included in GEM2010MAS65 clinical trial. We sequenced at an average read depth of 1500X and using Ion Proton sequencer (Thermo Fisher, USA, PaloAlto, CA). We used Ion Reporter software applying in-house modifications in call variants process. We also sequenced DNA from CD138 negative population in 30% of patients in order to filter out potential artifacts and stablish conditions for excluding germline variants. We first fitted a cox-proportional hazard model to predict survival and a second approach using a penalized regression LASSO. We identified 137 gene mutations in the 80 patients analyzed, 54 mutations were predicted pathogenic by Shif and Poliphen and 65 have been described in COSMIC database. 85% of patients harbored at least 1 mutation. The most frequently mutated genes were KRAS (16%), DIS3 (15%), NRAS (13%), BRAF (6%), and TP53 (5%), accounting for the 54% of the total number of mutations. The most frequently mutated pathways were RAS and NFKB in 34% and 28% of patients respectively. Mutational allele frequency for KRAS, BRAF and TP53 was, in all cases, lower than 50% while DIS3 showed mutations in a broad range (from 2 to 85%). Only one patient presented one mutation in NRAS at 73 % of allele frequency. For patients with more than one mutation, two different scenarios have been found. Some patients showed several genes mutated in a similar allele frequency values. On the contrary, a complex subclonal structure was confirmed in 3 patients, with mutations at very different clonal frequency. CD138 negative fraction was sequenced to confirm that these mutations were exclusive of plasma cells. The first of them had two mutations in DIS3 at 8 and 53% of allele frequency, second in KRAS and DIS3 at 13 and 63% respectively and the third in NRAS and IFNGR2 at 5 and 37% respectively. We found no differences in the survival based on pathway, mutational allele frequency or number of mutations. We also investigated pairwise associations between events and we not found significant correlations (Figure 1). Cox-proportional hazard model only showed a negative impact in survival of patients with TP53 mutations. This ultra-deep targeted sequencing strategy is able to detect mutations in most of patients, at very low allele frequency and using a minimal amount of DNA. Despite of its huge capacity to detect mutations, we have not identified new prognostic factors in MM patients treated with highly efficient therapies. However, this study again confirms the complexity of the genomic landscape of MM and the great heterogeneity between patients. The role of theses mutations at relapse as well as in the subsequent treatment effectiveness remains unknown and should be preferably investigated in larger cohorts of homogeneously treated MM patients. Figure 1 Figure 1. Disclosures Martínez-López: Novartis: Honoraria, Speakers Bureau. Oriol:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Stewart:celgene: Consultancy.

Abstract Previous work in our laboratory has identified the importance of the NFB signaling pathway for plasma cell (PC) survival and accumulation in Multiple Myeloma (MM). Although up to 43% of patient PCs appear to have activated NFkB by gene expression profiling (GEP), the remainder must rely on an alternate signaling pathway for their survival. We examined our MM GEP datasets and supervised the clustering of patients according to an index of NFkB activity. The majority of patients not activating NFkB were associated with a high IL-6/Stat3 signature, which included the protein phosphatase of regenerating liver-3 (PRL-3). Interestingly, analysis of the PRL-3 promoter revealed competing binding sites for both NFkB and STAT3 within the first intron, suggesting that these play a role in the transcriptional regulation of this gene. PRL-3 has been previously demonstrated to be absent in most normal tissues while its expression in MM cells is quite elevated. As normal PCs have low levels of PRL-3 expression, we determined the upper limit of normal to be 3SD above the mean of these cells. Using this cutoff we report that elevated PRL-3 expression is observed in plasma cells from 1/15 normal individuals, 4/22 MGUS patients and 47/126 patients with either smoldering or overt MM (6.7%, 18.2% and 45.6% respectively). Less than 10% of patients were seen to have both an elevated NFkB index and high PRL-3 expression. Patients with higher expression of PRL-3 were found to have statistically significant higher mean values of creatinine, C-reactive protein, lower albumin levels and a lower overall survival in our Mayo Clinic MM dataset. To examine the role PRL-3 in MM biology we first stimulated MM cell lines with IL-6 and observed PRL-3 up-regulation in two cell lines (INA6, OPM1) but not in JK6L (an NFkB activated cell line). Using a luciferase reporter we have determined that STAT3 induction of PRL3 expression requires the first intron of the PRL3 gene which contains the putative STAT3 binding site. Finally we show that PRL-3 inhibition by shRNA leads to decreased cell growth and loss of IL-6 responsiveness in vitro in at least one cell line. Based on these results, we propose that the majority of MM patients depend on either the NFkB or STAT3 signaling pathways for their survival and that these are to a large degree mutually exclusive. Our experimental results suggest that PRL-3 plays a role in IL-6/STAT3 signaling and that it is a promising target for drug development.