Key Points• A high frequency of RAS/RAF mutations and recurrent mutations in PDGFRA and JAK3 were found in relapsed multiple myeloma patients.• Patients with NRAS, but not KRAS, mutation exhibited significantly reduced sensitivity to bortezomib but not high-dose dexamethasone.Various translocations and mutations have been identified in myeloma, and certain aberrations, such as t(4;14) and del17, are linked with disease prognosis. To investigate mutational prevalence in myeloma and associations between mutations and patient outcomes, we tested a panel of 41 known oncogenes and tumor suppressor genes in tumor samples from 133 relapsed myeloma patients participating in phase 2 or 3 clinical trials of bortezomib. DNA mutations were identified in 14 genes. BRAF as well as RAS genes were mutated in a large proportion of cases (45.9%) and these mutations were mutually exclusive. New recurrent mutations were also identified, including in the PDGFRA and JAK3 genes. NRAS mutations were associated with a significantly lower response rate to single-agent bortezomib (7% vs 53% in patients with mutant vs wildtype NRAS, P 5 .00116, Bonferroni-corrected P 5 .016), as well as shorter time to progression in bortezomib-treated patients (P 5 .0058, Bonferroni-corrected P 5 .012). However, NRAS mutation did not impact outcome in patients treated with high-dose dexamethasone. KRAS mutation did not reduce sensitivity to bortezomib or dexamethasone. These findings identify a significant clinical impact of NRAS mutation in myeloma and demonstrate a clear example of functional differences between the KRAS and NRAS oncogenes. (Blood. 2014; 123(5):632-639)

Variations within proteasome ␤ (PSMB) genes, which encode the ␤ subunits of the 20S proteasome, may affect proteasome function, assembly, and/or binding of proteasome inhibitors. To investigate the potential association between PSMB gene variants and treatment-emergent resistance to bortezomib and/or long-term outcomes, in the present study, PSMB gene sequence variation was characterized in tumor DNA samples from patients who participated in the phase 3 Assess- IntroductionThe 20S core of the 26S proteasome degrades polyubiquitinated intracellular proteins 1 and is composed of 4 stacked rings, 1-4 each with 7 ␣ and 7 ␤ subunits. Three constitutive proteasome ␤ (PSMB) subunits, ␤5, ␤2, and ␤1 (encoded by the PSMB5, PSMB7, and PSMB6 genes, respectively), 5 are responsible for chymotrypsinlike, trypsin-like, and post-glutamyl peptide hydrolyzing activities, respectively. 6-8 On IFN-␥ stimulation, constitutive ␤ subunits are replaced by the IFN-inducible subunits ␤5i, ␤2i, and ␤1i (encoded by PSMB8, PSMB10, and PSMB9 genes, respectively) 5 to form the immunoproteasome. 6,9 Variations in PSMB subunits could potentially affect proteasome structure, assembly, function, and/or binding of proteasome inhibitors.Bortezomib (trade name Velcade; Millennium Pharmaceuticals) is approved in the United States and Europe for the treatment of patients with multiple myeloma (MM), 10,11 and in the United States for patients with relapsed mantle cell lymphoma. 10 Bortezomib selectively binds to the ␤5 subunit, leading to full inhibition of ubiquitinated protein hydrolysis. 7 In addition, bortezomib interacts with the ␤1 subunit 8 and, when bound to the ␤5 subunit in the chymotryptic catalytic site, is in close proximity to the ␤6 subunit. 12 Several studies have shown that PSMB5 variants can arise in vitro when tumor cell lines are cultured with bortezomib [13][14][15][16][17][18] ; it remains unclear whether this mechanism is relevant for bortezomib resistance in the clinical setting.The present study addressed whether variations in PSMB genes affect treatment-emergent resistance in bortezomib-treated MM patients or long-term outcome in MM patients. Sequence variation was characterized in coding regions of PSMB genes in pre-and posttreatment samples from patients who participated in the phase 3 Assessment of Proteasome Inhibition for Extending Remissions (APEX) trial of single-agent bortezomib versus high-dose dexamethasone (Dex) for the treatment of relapsed MM. 19 Study designReview boards at all participating institutions approved the (APEX) study, 19 and BM aspirates were obtained from consenting patients in accordance with the Declaration of Helsinki during the APEX trial. Tumor cells were purified and frozen for nucleic acid isolation as described previously. 20 Matching germline DNA samples were not collected. DNA samples were amplified using the QIAGEN REPLI-g whole genome amplification kit and For personal use only. on May 11, 2018. by guest www.bloodjournal.org From used for PCR reactions with primers for coding regions o...

BackgroundTAK733 is a novel allosteric, non-ATP-binding, inhibitor of the BRAF substrates MEK-1/2.MethodsThe growth inhibitory effects of TAK733 were assessed in a panel of 27 cutaneous and five uveal melanoma cell lines genotyped for driver oncogenic mutations. Flow cytometry, Western blots and metabolic tracer uptake assays were used to characterize the changes induced by exposure to TAK733.ResultsFourteen cutaneous melanoma cell lines with different driver mutations were sensitive to the antiproliferative effects of TAK733, with a higher proportion of BRAFV600E mutant cell lines being highly sensitive with IC50s below 1 nM. The five uveal melanoma cell lines had GNAQ or GNA11 mutations and were either moderately or highly sensitive to TAK733. The tested cell lines wild type for NRAS, BRAF, GNAQ and GNA11 driver mutations were moderately to highly resistant to TAK733. TAK733 led to a decrease in pERK and G1 arrest in most of these melanoma cell lines regardless of their origin, driver oncogenic mutations and in vitro sensitivity to TAK733. MEK inhibition resulted in increase in pMEK more prominently in NRASQ61L mutant and GNAQ mutant cell lines than in BRAFV600E mutant cell lines. Uptake of the metabolic tracers FDG and FLT was inhibited by TAK733 in a manner that closely paralleled the in vitro sensitivity assays.ConclusionsThe MEK inhibitor TAK733 has antitumor properties in melanoma cell lines with different oncogenic mutations and these effects could be detectable by differential metabolic tracer uptake.

Background & Aims Firmicutes bacteria produce metabolites that maintain the intestinal barrier and mucosal immunity. Firmicutes are reduced in the intestinal microbiota of patients with ulcerative colitis (UC). In a phase 1b trial of patients with UC, we evaluated the safety and efficacy of SER-287, an oral formulation of Firmicutes spores, and the effects of vancomycin preconditioning on expansion (engraftment) of SER-287 species in the colon. Methods We conducted a double-blind trial of SER-287 in 58 adults with active mild-to-moderate UC (modified Mayo scores 4–10, endoscopic subscores ≥1). Participants received 6 days of preconditioning with oral vancomycin (125 mg, 4 times daily) or placebo followed by 8 weeks of oral SER-287 or placebo. Patients were randomly assigned (2:3:3:3) to groups that received placebo followed by either placebo or SER-287 once weekly, or vancomycin followed by SER-287 once weekly or SER-287 once daily. Clinical endpoints included safety and clinical remission (modified Mayo score ≤ 2; endoscopic subscores 0 or 1). Microbiome endpoints included SER-287 engraftment (dose species detected in stool after, but not before, SER-287 administration). Engraftment of SER-287 and changes in microbiome composition and associated metabolites were measured by analyses of stool specimens collected at baseline, after preconditioning, and during and 4 weeks after administration of SER-287 or placebo. Results Proportions of patients with adverse events did not differ significantly among groups. A higher proportion of patients in the vancomycin/SER-287 daily group (40%) achieved clinical remission at week 8 than patients in the placebo/placebo group (0), placebo/SER-287 weekly group (13.3%), or vancomycin/SER-287 weekly group (17.7%) ( P =.024 for vancomycin/SER-287 daily vs placebo/placebo). By day 7, higher numbers of SER-287 dose species were detected in stool samples from all SER-287 groups compared with the placebo group ( P <.05), but this difference was not maintained beyond day 7 in the placebo/SER-287 weekly group. In the vancomycin groups, a greater number of dose species were detected in stool collected on day 10 and all subsequent time points, through 4 weeks post-dosing, compared with the placebo group ( P <.05). A higher number of SER-287 dose species were detected in stool samples on days 7 and 10 from subjects who received daily vs weekly SER-287 doses ( P <.05). Changes in fecal microbiome composition and metabolites were associated with both vancomycin/SER-287 groups. Conclusions In this small phase 1b trial of limited duration, the safety and tolerability of SER-287 were similar to placebo. SER-287 following vancomycin was significantly more effective than placebo for induction of remission in patients with active mild-to-moderate UC. Engraftment of dose species was facilita...

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3724 Poster Board III-660 Lymphomas account for about 5% of all cases of cancer in the US. In 2009, it is estimated that 74490 Americans will be diagnosed with lymphoma, which includes approximately 8510 new cases of Hodgkin's lymphoma and 65980 new cases of non-Hodgkin's lymphoma (NHL). An estimated 20790 men and women will die from lymphoma in 2009, the majority (93.8%) of which from NHL. Standard treatment includes chemotherapy and radiation to destroy the malignant lymphoma cells; however, newer therapies are currently being explored. Bortezomib is approved for multiple myeloma and relapsed mantle cell lymphoma, and is under clinical investigation in other types of NHL and other B-cell malignancies. Similar to bortezomib, MLN9708 is a modified dipeptidyl boronic acid that is a potent, reversible and specific inhibitor of the proteasome. MLN9708 preferentially binds to and inhibits b5 enzymatic activity of the 20S catalytic core. It is currently in Phase I trials for the treatment of solid and hematologic malignancies. MLN9708 immediately hydrolyzes to MLN2238, the biologically active form, on exposure to aqueous solutions or plasma. MLN2238 was used for all preclinical studies described below. We evaluated the ability of MLN2238 to inhibit tumor growth in two preclinical models of diffuse large B-cell lymphoma (DLBCL), an aggressive lymphoma that accounts for ∼40% of lymphomas in adults. OCI-LY10 and PHTX-22L are two examples of ABC-subtype of DLBCL. The antitumor activity of MLN2238 was evaluated in CB-17 Scid female mice bearing OCI-LY10 lymphoma xenografts. Tumor inhibition was determined by calculating the treatment over control (T/C) ratio of the mean tumor volume and the percentage of tumor growth inhibition (TGI) on the last day of the study. MLN2238 administered intravenous (IV) at 18 mg/kg once weekly (QW) resulted in tumor regression in 6 of 7 animals (T/C = 0.12; TGI = 88%) and a partial response in the remaining animal (43% reduction in tumor volume). MLN2238 administered at 8 mg/kg or 4 mg/kg IV QW resulted in a T/C of 0.42 and 0.36. Proteasome activity in the tumor was inhibited 49 and 44% 1 hour after a single IV dose of MLN2238 at 14 or 7 mg/kg, respectively. The other tumor model, PHTX-22L, is a primary tumor xenograft that was derived from a tumor surgically removed from the right lymph node of a 71-year-old Caucasian male. SCID NOD female mice implanted with PHTX-22L tumor fragments were treated twice weekly (BIW) with IV doses of vehicle, MLN2238 at 4 mg/kg SC once daily (QD), MLN2238 at 14 mg/kg IV BIW or bortezomib at 0.8 mg/kg IV BIW. The strongest antitumor activity was seen in PHTX-22L tumor bearing mice treated with IV MLN2238 14 mg/kg BIW (T/C = 0.14; TGI = 86%, p < 0.001) or SC MLN2238 4 mg/kg QD (T/C = 0.15; TGI = 85%, p < 0.01). These dosing regimens significantly inhibited tumor growth compared with vehicle treatment. In the same study, bortezomib treatment IV at 0.8 mg/kg on a BIW schedule had no antitumor effect (T/C = 0.88; TGI = 12%, p = 0.86) and was not statistically different from the vehicle treated group. Proteasome activity was significantly inhibited in both blood and tumor following a single dose of MLN2238 administered IV at 14 mg/kg or SC at 4 mg/kg. Proteasome activity was inhibited greater than 50% for up to 8 hrs following the IV dose in both blood and tumor, whereas proteasome activity recovered more quickly following the SC dose. In a separate preclinical study, the antitumor activity of MLN2238 administered at lower doses and on alternate schedules was examined. SCID NOD mice bearing PHTX-22L xenografts were administered MLN2238 IV at 11 mg/kg BIW, 7 mg/kg BIW, 11 mg/kg QW or 5 mg/kg QDx2/week resulted in significant antitumor activity (T/C = 0.04, 0.08, 0.15, 0.16; TGI = 96, 92, 85, 84%, respectively). More importantly, drug treatment caused tumor regression (tumor volume at the end of the study was less than 50% of the starting tumor volume) in 8 of 10 mice in the 11 mg/kg BIW, 5/10 in the 7 mg/kg BIW, 2/8 in the 11 mg/kg QW and 1/10 in the 5 mg/kg QDx2/week treatment groups. The efficacy of MLN2238 dosed IV either at 7 mg/kg QW or 3.5 mg/kg BIW in this model was less efficacious (T/C = 0.61 and 0.64). In addition to PHTX-22L, we have access to several other primary lymphoma tumor xenograft models that we plan to evaluate for sensitivity to MLN2238 treatment. These studies demonstrate that MLN2238 is active in preclinical models of lymphoma, and that MLN2238 has antitumor activity in a model of lymphoma that is refractory to bortezomib treatment. Disclosures: Donelan: Milllennium: Employment. Bannerman:Milllennium: Employment. Bano:Milllennium: Employment. Babcock:Milllennium: Employment. Hales:Millennium Pharmaceuticals: Employment. Stringer:Milllennium: Employment. Burke:Milllennium: Employment. Danaee:Milllennium: Employment, Equity Ownership. Faron-Yowe:Milllennium: Employment. Koenig:Milllennium: Employment. Lichter:Milllennium: Employment. Kupperman:Milllennium: Employment.

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