Citations

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.

Abstract Background: Proteasome inhibition with carfilzomib (CFZ) has shown to be effective therapy in relapsed refractory multiple myeloma (MM).1 Cardiac toxicity induced by CFZ has been reported in a small subset of patients treated in phase 3 clinical trials, including congestive heart failure (CHF) in approximately 2-5% of patients. The natural history or reversibility of cardiac dysfunction with CFZ has not been fully reported. The myocardium constantly contracts in adults and thus, adequate protein metabolism may be essential; and hence, effective proteasome degradation may pose a risk for contractility. Methods and results: We identified 12 patients, who received CFZ at Mayo Clinic in Arizona, and had a drop in ejection fraction (EF) attributable to the use of CFZ. Medical records were reviewed for baseline cardiovascular (CV) risk factors, CV disease reversibility and mortality resulting from CFZ induced cardiac dysfunction. Patients who had a drop in EF attributed to other causes, were not included in this count. The median age at the time of initiation of therapy with CFZ was 62.5 years (range, 55-70 years), median body mass index was 27.65 (range, 21 - 40), with 4 (33%) patients being obese and 6 (50%) in the overweight range. Two (17%) patients had history of coronary artery disease while 9 (75%) had hypertension, and were on medical treatment prior to CFZ initiation. Six (46%) patients had a previous history of CHF. Two of these had symptomatic systolic dysfunction, however at the time of CFZ initiation, EF had improved. The other 4 with prior CHF had sub-normal EFs noted on echocardiograms done prior to initiation of CFZ (median EF of these 4 was 49%). Overall, baseline EF was available for 10 out of these 12 patients and median EF at baseline was 55% (range, 45 - 67%). Other risk factors predisposing to cardiac dysfunction were smoking in 1 patient and history of radiation to the chest in 2 (17%) patients. No patient had received cardiotoxic dose of anthracyclines prior to starting CFZ. Myeloma therapies: All patients had received multiple lines of therapy for MM (median 4, range, 2-7). Ten (83%) patients had previous received bortezomib and 10 (83%) had received autologous stem cell transplant. Cardiac events and CFZ use: Median duration of CFZ therapy received prior to documented systolic dysfunction was 4 months (range, 0.5 - 20 months), with 3 episodes happening with less than one month of total CFZ therapy. Five out of the 12 (42%) patients had previously discontinued CFZ for other reasons when the systolic dysfunction was noted. However they were included since CHF occurred shortly after treatment (median of 2 months, range, 0.5 - 3). The median drop in EF was 22% (range, 9 - 45%). Recovery/ outcomes: Follow-up echocardiograms were available in 8 patients and all of these showed some improvement in EF (table 1). Median improvement in EF noted by the time of last follow up was 16% (range, 7-30%). Median best EF noted after the initial drop was 48% (range, 35 - 67%). Two patients never discontinued CFZ and EF improved spontaneously, to 67% and 64% respectively, after an initial drop. Another patient was rechallenged with CFZ due to progression of MM, 8 months after initial discontinuation, and did not have recurrence of systolic dysfunction. No patients succumbed to CHF. Six (50%) out of these 12 patients had died by the time of last follow-up. Cause of death was progressive MM in 4 (67%) and sepsis secondary to immunosuppression resulting from chemotherapy in 2 (33%). Conclusions: Our data, although with limited number of patients, suggests using caution when treating patients who have underlying cardiac risk factors with CFZ. Nevertheless, the cardiotoxicity was mostly reversible, with the minimal EF after recovery of 35%. In the setting of a life threatening malignancy, the impact and reversibility of cardiotoxicity needs to be understood so as not to prevent the utilization of otherwise effective therapy such as CFZ. Larger studies are needed to confirm these findings. References: 1. Stewart et al. Carfilzomib, Lenalidomide and Dexamethasone for relapsed multiple myeloma. NEJM 2015;372:142-52. Disclosures Mikhael: Celgene: Research Funding; Abbvie: Research Funding; Sanofi: Research Funding; Onyx: Research Funding. Reeder:Novartis, Millennium, BMS and Celgene: Research Funding. Bergsagel:Amgen, BMS, Novartis, Incyte: Consultancy; Novartis: Research Funding. Fonseca:Millennium, a Takeda Company: Consultancy; Sanofi: Consultancy; AMGEN: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Millennium, a Takeda Company: Consultancy; Novartis: Consultancy; AMGEN: Consultancy; Janssen: Consultancy; Janssen: Consultancy; BMS: Consultancy; Millennium, a Takeda Company: Consultancy; Bayer: Consultancy; Millennium, a Takeda Company: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Celgene: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; AMGEN: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; 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; Celgene: Consultancy; BMS: Consultancy; Bayer: Consultancy; Novartis: Consultancy; Sanofi: Consultancy; AMGEN: Consultancy.

Abstract Background: Pomalidomide has demonstrated excellent activity in patients with relapsed, lenalidomide refractory, multiple myeloma (MM). Between November 2007 and March 2012, we enrolled 285 patients with relapsed MM on 5 sequential phase 2 trials; patients received pomalidomide at 2mg or 4 mg daily with weekly dexamethasone (Pom/dex). The approved dose of pomalidomide is 4 mg for 21 of 28 days. We wished to compare efficacy, tolerability and long-term outcomes between cohorts treated with 2 mg or 4mg daily continuously and 4mg daily for 21/28 days. Methods: After excluding two ineligible patients, 283 patients with lenalidomide refractory, relapsed MM from 5 sequential cohorts were analyzed. These patients were divided into 3 groups: Group1 received Pom 2mg for 28/28 day cycle (N= 69), Group 2 received Pom 4 mg for 28/28 day cycle (N= 95) and Group 3 received Pom 4mg for 21/28 day cycle (N= 119). All patients received oral dexamethasone given 40 mg daily on days 1, 8, 15, and 22. Response was assessed by the IMWG Uniform Response criteria. All patients received aspirin 325 mg daily for DVT prophylaxis or full dose anticoagulation. Results: The median age was 63 years (32-85); 35% were female. The median time from diagnosis was 53 months and the median number of prior regimens was 4. 127 (46%) had high-risk molecular markers. Prior therapies (% received) included lenalidomide (100%), thalidomide (46%), bortezomib (78%), autologous stem cell transplant (71%), and allogeneic transplant (4%). The median follow-up is 16.4 months (3.2-64.4). Forty eight percent are alive and 26% remain progression free; 15 patients are continuing to receive treatment. Frequency of AEs by groups are shown in Table 1. The most notable difference is grade 3+ neutropenia seen in 39% of group 1 and 56% and 57% of groups 2 and 3. Confirmed responses of PR or better were seen in 29% (group1), 35% (group2) and 24% (group3). Median duration of response (DOR) was 14.1 months (group1), 14.5 months (group2) and 10.2 months (group3). Median PFS was 5.5 months (group1), 6.9 months (group2) and 4.3 months (group3). Although the dose level cohorts were sequential rather than randomized, we compared OS between the dose levels in an exploratory manner. There was no significant difference in OS between dose levels (p=0.26). Median overall survival (OS) was 16.6 months (group1), 21.9 months (group2) and 16.0 months (group3). Conclusions: Pom/dex is active and well tolerated even in heavily pretreated patients Responses are durable. Response rates and overall toxicity are similar between the 2 mg and 4 mg doses. Neutropenia is more common in those receiving doses of 4mg daily or for 21/28 days compared to those receiving 2 mg daily. Table 1. All Grades Grade 3+ 2mg 28 Day 4mg 28 Day 4mg 21 Day 2mg 28 Day 4mg 28 Day 4mg 21 Day Anemia 68% 58% 74% 14% 15% 27% Lymphopenia 22% 51% 11% 16% 32% 8% Neutropenia 71% 82% 77% 39% 57% 56% Thrombocytopenia 51% 61% 63% 10% 9% 23% Leukopenia 59% 77% 72% 26% 38% 39% Pneumonia 7% 11% 12% 6% 7% 11% Fatigue 51% 65% 60% 9% 5% 8% Neuropathy 28% 32% 28% 0% 3% 0% Elevated Blood Glucose 10% 21% 8% 4% 6% 3% Pneumonitis 3% 2% 3% 3% 1% 1% VTE (Thrombosis) 3% 3% 3% 1% 3% 3% Secondary Malignancy 0% 2% 1% 0% 2% 1% Figure1. Kaplan Meier Overall Survival Curves Figure1. Kaplan Meier Overall Survival Curves Disclosures Lacy: Celgene: Research Funding. Fonseca:Medtronic, Otsuka, Celgene, Genzyme, BMS, Lilly, Onyx, Binding Site, Millennium, AMGEN: Consultancy, patent for the prognostication of MM based on genetic categorization of the disease. He also has sponsored research from Cylene and Onyx Other, Research Funding. Bergsagel:Novartis: Research Funding; Constellation Pharmaceutical: Research Funding; OncoEthix: Research Funding; MundiPharma: Research Funding. Stewart:Novartis: Consultancy; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Array BioPharma: Consultancy; Sanofi: Consultancy; Takeda Pharmaceuticals International Co.: Research Funding. Reeder:Millennium, Celgene, Novartis: Research Funding. Mikhael:Onyx: Research Funding; Celgene: Research Funding; Sanofi: Research Funding; Novartis: Research Funding.

Abstract Introduction - With over 12,000 deaths from MM anticipated in 2016, nearly all patients (pts) with multiple myeloma (MM) will become "quad refractory" to IMIDs (lenalidomide and pomalidomide) and proteasome inhibitors (bortezomib and carfilzomib), and eventually "penta refractory" to anti-CD38 Abs (daratumumab and isatuximab), defining high unmet need populations. Selinexor, an oral selective XPO1 inhibitor, induces nuclear accumulation and activation of tumor suppressor proteins, inhibition of NF-kB, and inhibition of translation of several oncoprotein mRNAs such as c-myc and cyclin D. Selinexor showed potent induction of apoptosis of MM cells independent of p53 signaling. In phase 1 clinical studies, selinexor with low dose dexamethasone (Sd) demonstrated potent anti MM activity in pts with MM. Methods - This phase II clinical trial evaluated Sd in pts with MM refractory to bortezomib, carfilzomib, lenalidomide, and pomalidomide ("quad"), with a subset also refractory to an anti-CD38 Ab ("penta"). Inclusion required CrCL≥20 mL/min, ANC≥1000/µL, platelets ≥50K/µL (≥30K if plasma cells were ≥50% of marrow cellularity). Pts were treated twice weekly (BIW) with oral selinexor 80 mg for 6 or 8 doses per 28 day cycle and dexamethasone (dex) 20 mg BIW. All pts received 5-HT3 antagonists. The primary objective was to determine the overall response rate (ORR) per IMWG criteria and duration of response (DOR), both adjudicated by an independent review committee (IRC). Secondary endpoints include progression free survival (PFS) and overall survival (OS). FISH analyses and gene expression profiling were performed on bone marrow aspirates. Results - 79 pts were enrolled: 48 quad (24 M/24 F, median age 62 yrs) and 31 penta (13 M / 18 F, median age 68 yrs). Both groups had a median of 7 prior treatment regimens including multiple dex-containing regimens. Baseline laboratory abnormalities included grade (Gr)≥3 anemia in 13% and Gr≥3 thrombocytopenia in 8%. Most penta pts received 8 doses / cycle (65%); most quad pts received 6 doses / cycle (83%). Common treatment-related adverse events (TRAEs) hematological: thrombocytopenia (72%, Gr 3/4 58%), anemia (48%, Gr 3 25%) and neutropenia (29%, Gr≥3 21%). TRAEs non-hematological: nausea (72%, Gr 3 6%), fatigue (62%, Gr 3 14%) anorexia (49%, Gr 3 3%), vomiting (43%, Gr3 4%), asymptomatic hyponatremia (42%. Gr 3 20%), diarrhea (42%, Gr 3 5%) and weight loss (33%, Gr 3 1%). There was one case of febrile neutropenia (1%) and one case of clinically significant bleeding related to thrombocytopenia (1%). Seventy pts have discontinued therapy: PD (73%), AEs (17%), physician/pt preference (1%) and 6 deaths (one case related to selinexor, intracranial bleed in pt with Gr4 thrombocytopenia). Nine pts remain on study. Efficacy was evaluated in 78 pts (1 pt did not have measurable disease). The IRC-determined ORR (≥PR) for all pts was 21%, including 5% VGPR. ORR was 21% for quad pts and 20% for penta pts. Clinical benefit rates (≥MR) were 32% (all), 29% (quad), and 37% (penta). Median OS was 9.3 months (mo) for all pts, >11 mo (median not reached) for responders (≥PR), and 5.7 mo for non-responders. Median DOR in responding pts was 5 mo, and median PFS in all pts was 2.1 mo. Baseline cytogenetics were assessed in 41 pts. The ORR in 18 pts with high-risk FISH abnormalities was 33% (Table 1). Notably, 3 of the 13 pts with a 17p abnormality responded (ORR 23%). Transcriptomic profiling revealed differentially expressed genes (DEGs) between responders and non-responders in both whole blood RNA and CD138+ bone marrow cells. Pathways enriched in responders included IL-6, IL-8 and IGF-1 pathways. Conclusions - Oral Sd is active in heavily pretreated pts with refractory MM, including those with MM refractory to anti-CD38 Ab and those with high-risk cytogenetic abnormalities. Response was associated with longer survival. The main toxicities of Sd are thrombocytopenia, nausea, anorexia, and fatigue. AEs were manageable with supportive care and dose interruptions/reductions. To our knowledge, this is the first report of anti-tumor activity in the penta-refractory MM population. This population of MM pts has exhausted all currently available treatment options and has an extremely poor prognosis and therefore requires new therapies. Expansion of this trial in this high unmet medical need, penta refractory population is planned. Table 1 Activity of Sd in Patients with High Risk MM Cytogenetics Table 1. Activity of Sd in Patients with High Risk MM Cytogenetics Disclosures Vogl: Constellation: Research Funding; Karyopharm: Consultancy; Acetylon: Research Funding; GSK: Research Funding; Calithera: Research Funding; Teva: Consultancy; Celgene: Consultancy; Takeda: Consultancy, Research Funding. Jagannath:Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Merck: Consultancy; Celgene: Consultancy. Baz:Bristol-Myers Squibb: Research Funding; Takeda/Millennium: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Signal Genetics: Research Funding; Novartis: Research Funding; Karyopharm: Research Funding; Merck: Research Funding. Nooka:Spectrum, Novartis, Onyx pharmaceuticals: Consultancy. Richter:Takeda: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Janssen: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau. Vij:Karyopharm: Honoraria; Amgen: Honoraria, Research Funding; Celgene: Consultancy; Takeda: Honoraria, Research Funding; Novartis: Honoraria; Bristol-Myers Squibb: Honoraria; Janssen: Honoraria. Schiller:Incyte Corporation: Research Funding. Costa:Sanofi: Honoraria, Research Funding. Chari:Array Biopharma: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Research Funding; Amgen Inc.: Honoraria, Research Funding; Novartis: Consultancy, Research Funding. Siegel:Takeda: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Merck: Honoraria. Fonseca:Janssen: Consultancy; AMGEN: Consultancy; Millennium, a Takeda Company: Consultancy; Bayer: Consultancy; Sanofi: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; AMGEN: Consultancy; Novartis: Consultancy; Millennium, a Takeda Company: Consultancy; Sanofi: Consultancy; AMGEN: Consultancy; Janssen: Consultancy; BMS: Consultancy; Celgene: Consultancy; BMS: Consultancy; Millennium, a Takeda Company: Consultancy; Millennium, a Takeda Company: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Bayer: Consultancy; Novartis: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; AMGEN: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; 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; Celgene: Consultancy. Kauffman:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Saint-Martin:Karyopharm: Employment. Picklesimer:Karyopharm: Employment. Friedlander:Karyopharm Therapeutics: Employment. Choe-Juliak:Karyopharm Therapeutics: Employment.

Abstract Background: The proteasome inhibitor Bortezomib (BTZ) is an efficient treatment option for both, newly diagnosed and relapsed/refractory multiple myeloma (MM). Despite the effectiveness, most patients eventually acquire drug resistance for reasons not fully understood Materials and methods: To better understand BTZ resistance mechanism we used a CRISPR library (GeCKO V2) targeting 19052 human genes, trying to identify genes responsible for BTZ resistance. CRISPR sgRNAs targeting the ERN1-XBP1 pathway were used as positive controls. We first infected RPMI 8226 myeloma cell line expressing Cas9 with the CRISPR library packaged into lenti-vectors and selected for resistance to BTZ. Surviving cells were subjected to next generation sequencing. Based on the initial screen results we constructed a second CRISPR sgRNA library including 31 genes, each gene targeted with four sgRNAs. After the second round screening and subsequent sequencing, we selected the top 20 genes for individual validation. Results: Proteasome regulatory gene PSMC6 was identified as the only reproducible gene conferring BTZ resistance. Interestingly, the same gene was independently found by a second group using a CRISPR approach (Sheffer M et al. ASH Abstract 273, 2014). Resistance was reproducible using a PSMC6 knockout by three individual CRISPR sgRNAs targeting exonic regions and one pair of SgRNA targeting intron region flanking exon for the deletion of exon one. PSMC6 knockout was verified by PCR and Sanger sequencing. Sensitivity to BTZ was rescued by over expression of PSMC6 cDNA in RPMI 8226 cells harboring a deletion of PSMC6 exon 1. MM cells lacking PSMC6 also developed resistance against Carfilzomib. Resistance was reproduced on a second MM cell line, KMS11. We did not see any significant difference of toxicity in PSMC6 deleted cells for other chemicals tested (tunicamycin, staurosporine, dexamethasone and melphalan). We demonstrated that the sensitivity of chymotrypsin-like activity of proteasome against BTZ was significantly reduced in cells lacking PSMC6. Consequently, MM cells without the PSMC6 gene were relatively resistant to apoptosis induced by BTZ, which was verified by Western blot for caspase 8 degradation and luminescent assay for caspase 3/7 activities. Clinically we could not correlate the PSMC6 expression level with the outcome of BTZ treatment in BTZ naive patients using publically available gene expression data. We initially used CRISPR sgRNAs targeting ER stress pathway (ERN1 and XBP1) as a positive control. However we could not derive any resistant cells from the experiment. We also could not identify any sgRNAs targeting the ERN1-XBP1 pathway from our whole exome screen and next generation sequencing. Since this contradicts published data, we decided to knockout ERN1 and XBP1 genes individually. Clones of cells with successful knockout of ERN1 in three cell lines (RPMI 8226, KMS11 and JJN3) and XBP1 in two cell lines (RPMI 8226 and KMS11) were tested for response to BTZ and Carfilzomib , however we did not find any drug response difference between the knockout and parent cells. We also found that the ERN1-XBP1 knockout cells did not show difference in response to ERN1 specific inhibitors (4u8C and STF-038010) and ER stress inducers (tunicamycin and thapsigargin) compared to parental cells. Conclusions: Human multiple myeloma cells lacking the PSMC6 gene develop significant resistance to apoptosis induced by BTZ. We have however not found a correlation of PSMC6 expression levels with outcome to BTZ treatment in BTZ naïve patients. We are therefore currently investigating the PSMC6 mutation rate in relapsed MM patients after proteasome treatment. In contrast to previous reports showing that progenitor MM cells lacing XBP-1 or ERN-1 invoked BTZ resistance, we were not able to demonstrate a change in sensitivity after full CRISPR knock out of either ERN1 or XBP1. It has long been believed that ERN1-XBP1 pathway plays an important role for MM treatment, leading to the development of ERN1 specific inhibitors. However, we demonstrated that the toxicity of two ERN1-specific inhibitors appears independent of the ERN1-XBP1 pathway. We also demonstrated that the toxicity of two important ER stress inducers, tunicamycin and thapsigargin, is independent of the ERN1-XBP1 pathway. Disclosures Stewart: Celgene: Consultancy; Oncospire Inc.: Equity Ownership; BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Abstract Background: The current standard of care for eligible newly diagnosed multiple myeloma (MM) patients is induction therapy with novel agents followed by high dose chemotherapy and autologous stem cell transplant (ASCT). The International Myeloma Working Group proposed the Revised International Staging System (R-ISS) based on the presence of adverse chromosomal abnormalities (CA) detected by FISH (t(4;14), t(14;16) or del17p), in combination with ISS and LDH at diagnosis. However, there are a limited number of studies that have validated this risk model in the transplant and novel agents setting. Aims: To determine whether R-ISS is an appropriate risk-model for estimating overall survival (OS) and progression free survival (PFS) for transplant-eligible MM patients. Patients and Methods: We retrospectively studied a cohort of 519 MM patients who received novel drugs in the induction and subsequently underwent ASCT at Mayo Clinic Arizona from 2005 to 2014. In all, 95 patients met the inclusion criteria: comprising complete data at diagnosis (ISS, serum LDH level, and CA by FISH). The primary endpoint was OS from SCT and the secondary end point was PFS from ASCT. R-ISS groups were defined as described by Palumbo et al. J Clin Oncol. 2015; 33(26):2863-2869. Results: There were 50 (52.6%) men and 45 (47.4%) women who underwent ASCT in this period, with a median age at the time of transplant of 66-years-old (range, 36-78). There were 27 patients (28.4%) with high-risk CA: 12 patients (12.6%) with del17p; 11 patients (11.6%) with t(4;14); and 6 (6%) with t(14;16). In addition, 8 patients (8.5%) had high LDH levels and 9 patients (9.5%) presented with renal impairment at diagnosis. The patients were staged at diagnosis according to the three R-ISS groups: 44 patients (46.3%) had stage I, 26 (27.4%) had stage II, and 25 (26.3%) had stage III. CyBorD was the preferred induction regimen which was received by 42 patients (44%). There were 14 patients (15%) who received at least 2 lines of induction. All patients were in at least partial response (PR) at the moment of transplant: 32 in complete response (CR), 32 in very good partial response (VGPR) and the remaining 31 in PR. The response achieved at day +100 after ASCT improved, with 54 patients (57%) in CR, and only 18 patients (19%) in PR. After a median follow-up of 61 months (range, 14-135), median OS from SCT was 108 months (95% CI: 85 - 132 months) and the median PFS was 45.4 months (95% CI: 31.1 - 53.8 months) in the whole series.MM patients with R-ISS III had a significantly shorter median OS compared to patients with R-ISS II or R-ISS I (32.1 months vs. 94.7 months vs. not reached, respectively, P<0.0001) (Figure). No statistically significant differences in baseline characteristics were identified among these groups to explain the differences in OS observed. PFS among these groups was not statistically significant, only showing a trend towards shorter PFS in R-ISS III compared with either R-ISS I or II: median 22.1 months vs. 35.7 months, respectively, (P=0.2). Renal impairment at diagnosis, IgA subtype, ≥ 2 lines of induction treatment, and less than CR achieved at day +100 after ASCT were also associated with significantly inferior OS. Multivariate analysis selected R-ISS as an independent predictor for OS (HR: 2.3, 95% CI: 1.1-4.8; P=0.03), as well as ≥ 2 lines before ASCT. CR at day +100 after ASCT was the most important independent factor for predicting PFS (HR: 0.4; 95% CI: 0.2-0.6; P<0.001). Conclusion: R-ISS assessed at diagnosis was an independent predictor for OS after ASCT in our series, with median OS for the different R-ISS groups comparable to those reported by Palumbo et al. in their subgroup of younger patients. Thus, this study lends further support for the R-ISS as a reliable prognostic tool for estimating OS in transplant-eligible MM patients. In addition, new treatment approaches are needed for the high-risk patients (R-ISS III) with a median OS of 2.5 years. Figure Figure. Disclosures Reeder: Millennium: Research Funding; BMS: Research Funding; Celgene: Research Funding; Novartis: Research Funding. Mikhael:Abbvie: Research Funding; Onyx: Research Funding; Sanofi: Research Funding; Celgene: Research Funding. Bergsagel:Amgen, BMS, Novartis, Incyte: Consultancy; Novartis: Research Funding. Stewart:celgene: Consultancy. Fonseca:Janssen: Consultancy; Celgene: Consultancy; Sanofi: Consultancy; Novartis: Consultancy; Bayer: Consultancy; AMGEN: Consultancy; AMGEN: Consultancy; AMGEN: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; AMGEN: Consultancy; Millennium, a Takeda Company: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Novartis: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Millennium, a Takeda Company: Consultancy; Millennium, a Takeda Company: Consultancy; Bayer: Consultancy; Celgene: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; BMS: Consultancy; Millennium, a Takeda Company: Consultancy; BMS: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms.

Abstract Background: Pomalidomide is an immunomodulatory agent (IMiD®) that has been approved for treatment of relapsed and refractory multiple myeloma (MM). Combinations of IMiDs and proteasome inhibitors offer the potential for deeper and more durable responses due to enhanced efficacy. Preliminary results of a phase 1 study of twice weekly bortezomib with pomalidomide have been reported with promising results (Richardson, ASH 2012). This phase I/II trial was designed to evaluate the maximum tolerated doses (MTD) as well as safety and efficacy of the combination of pomalidomide, once weekly bortezomib and dexamethasone (PVD) in patients with relapsed, lenalidomide refractory, MM. Patients and methods: We included patients with relapsed MM who had 1-4 prior lines of therapy and were resistant or refractory to lenalidomide. In the phase I portion of the trial, dose level 1 consisted of pomalidomide 4 mg days 1-21 PO, bortezomib 1.0mg/m2 days 1,8,15, and 22 IV or SQ and dexamethasone 40 mg days 1,8,15, and 22 PO, given every 28 days. Bortezomib was increased to 1.3mg/m2 for dose level 2 and and was adopted for the phase 2 portion. The primary aim of the phase I cohort was to determine the MTD of the combination, and for the phase II cohort was to evaluate the confirmed response rate (PR, VGPR, or CR) in relapsed refractory MM. Response was assessed by the IMWG criteria and toxicity was graded using the CTCAE v4.0. Results: 50 patients were accrued between March 2012 and July 2014 (dose level (DL)1: 3, DL 2: 6, Phase II: 41). We describe results in 47 patients treated at MTD and phase II. Median age was 66, 51% were female and median time from diagnosis to study was 46 months (15-142). Twenty five percent had mSMART defined high-risk status. Median number of prior regimens was 3. All patients had prior lenalidomide, 68% had stem cell transplant, 17% received thalidomide, 56% had alkylators and 57% had bortezomib. With median follow up of 9 months, 72% remain progression free, 96% are alive and 66% remain on treatment. The most common AEs at least possibly attributable to the combination were anemia, fatigue, leukopenia and thrombocytopenia; however, the majority of these were grade 1-2. Grade ≥3 AEs (regardless of attribution) that occurred in at least 3 patients included neutropenia (29), leukopenia (15), lung infection (6), lymphopenia (8) dyspnea (3) and syncope (3). DVT/PE occurred in one patient. Among the 42 patients who were evaluable, confirmed responses (PR, VGPR, or CR) were seen in 34 (81%) including sCR (3), CR (5), VGPR (8), PR (18). Confirmed responses were seen in 9 of 11( 82%) high risk patients. Median progression free survival was 17.7 months (95%CI: 9.5-NA). Conclusions: PVD is a highly effective combination in patients refractory to lenalidomide with confirmed responses in over 80%. Weekly administration of bortezomib enhanced the tolerability and convenience of this regimen. Toxicities are manageable, mostly consisting of mild cytopenias with no significant neuropathy or DVT. PVD is a highly attractive option in patients with relapsed and refractory MM. Disclosures Lacy: Celgene: Research Funding. Lust:Senesco: PI Other. Fonseca:Medtronic, Otsuka, Celgene, Genzyme, BMS, Lilly, Onyx, Binding Site, Millennium, AMGEN: Consultancy, patent for the prognostication of MM based on genetic categorization of the disease. He also has sponsored research from Cylene and Onyx Other, Research Funding. Stewart:Celgene: Consultancy; Novartis: Consultancy; Array BioPharma: Consultancy; BMS: Consultancy; Millenium: Research Funding. Reeder:Millennium, Celgene, Novartis: Research Funding. Mikhael:Onyx: Research Funding; Celgene: Research Funding; Sanofi: Research Funding; Novartis: Research Funding.

Abstract Background: In Phase II trials, carfilzomib (Cfz) was associated with dyspnea (34%), hypertension (14%), renal insufficiency (24%), and peripheral edema (24%) with cardiac events reported in 7% of patients (pts). In order to better understand these toxicities, we prospectively instituted detailed cardiac evaluation in 62 Cfz treated multiple myeloma (MM) pts. Methods: Pts received Cfz between 8/11-5/14. Parameters recorded included Cfz dose, concurrent chemotherapy, hydration, blood pressure, day 1 and 2 creatinine, troponin and NT-proBNP, baseline and cycle 4 echocardiograms with EF, strain and compliance. Notable cardiac events were examined for attribution. Results: Median age was 65 years, with 60% male. 20 pts had no prior therapies, 42 were relapsed; mean 4 prior therapies (range 1-10). 20 received induction chemotherapy (Cfz, dex, thalidomide, cyclophosphamide). At relapse, Cfz was given alone in 21 pts, with added cyclophosphamide in 10, and with an IMiD in 10. Cfz dose was 20mg/m2 days 1,2 and ranged from 27-45mg/m2in subsequent treatment days. Dex was given at 20-40mg weekly in 77% pts. Hydration (250-500ml) was delivered in 89% of patients pre and 63% post treatment. Baseline hypertension (>140mmHg) was present in 20% of pts and rose ≥10mmHg in 26% on day 2, but dropped ≥10mmHg in 36%. On day 8, more pts (32%) had hypertension pre-treatment. Baseline creatinine was elevated in 25% pts. 7 of 55 (13%) pts had a creatinine rise (≥0.3 mg/dL) on day 2, 5 of whom had elevation at baseline. Thus, with normal baseline creatinine and pre-treatment hydration 2/45 (4%) pts had a creatinine rise. Troponin levels on day 1 were normal in all 16 tested patients. 2/25 pts had an elevated troponin on day 2, one with associated MI on day 4. In these 2 pts, symptomatic pre-existing ischemic heart disease and severe cardiac amyloid were confounding factors. Mean baseline NT-proBNP (n=22) was elevated at 565 pg/mL (range <50-3666; normal <77) with normal baseline values in only 41%. On day 2 (n=25), the mean was significantly higher at 1580 pg/mL. Between day 1 and 2, 72% of pts had a rise ≥50 pg/mL; mean change 1387 (range 0 to +6187). On day 2, 52% had a NT-proBNP ≥500 pg/mL, while 36% were ≥1000 pg/mL. 30 pts had baseline and post-C4 treatment echocardiograms, all with normal pre-treatment EFs. Three pts (10%) developed impaired systolic function (EF<50%), however, 2 had plausible concurrent alternative etiologies (VTE/PE, hospitalized multilobar pneumonia) while one patient (during C2) had an asymptomatic unexplained decline in EF (56 to 42%) detected pre-op while hospitalized for hip fracture. Thus, 1/30 (3%) had an EF change in absence of concurrent acute illness. A fourth patient had normal echo at C4 but was hospitalized at C10 with severe sepsis and a diminished EF was documented. All 4 pts with low EF were pre-treated (≥2 therapies plus SCT). Changes in cardiac strain often precede an EF fall. Only 14/25 pts had normal baseline cardiac strain (≥ -18%). Post treatment strain measurement significantly improved in 4 pts and worsened in 4 pts, the latter accompanied by EF decline within the normal range in 2 pts (68/60%, 65/58%). 28 pts had E/e’ ratio (LVEDP) reviewed as a marker of ventricular compliance. 46% had abnormal ratios (≥ 15), prior to Cfz treatment. 20% of patients experienced worsening parameters post treatment, however, an equal number of pts demonstrated improvement. There was no consistent correlation with change in EF. Conclusions: MM pts frequently have baseline elevated cardiac peptides (59%) and abnormal cardiac strain (15% of newly diagnosed and 36% at relapse). A frequent, and sometimes dramatic, rise in the NT-proBNP occurs immediately following Cfz based chemotherapy. Acute structural cardiac events were however uncommon (3%) in the absence of confounding illness. Overall 5/62 (8%) patients had serious cardiac events during treatment with Cfz (4 EF decline, 1 MI). These toxicities were considered probably attributable to Cfz in 3/62 (5%) patients. Prospective controlled studies with longer term follow up and bortezomib treated controls are necessary to accurately document the cardiovascular impact of Cfz and the role of proteasome inhibition. Disclosures Off Label Use: bortezomib was used in combination therapy to treat relapsed low grade lymphomas and Waldenstrom's macroglobulinemia. Fonseca:Medtronic, Otsuka, Celgene, Genzyme, BMS, Lilly, Onyx, Binding Site, Millennium, AMGEN: Consultancy, patent for the prognostication of MM based on genetic categorization of the disease. He also has sponsored research from Cylene and Onyx Other, Research Funding. Bergsagel:Novartis: Research Funding; Constellation Pharmaceutical: Research Funding; OncoEthix: Research Funding; MundiPharma: Research Funding. Reeder:Millennium, Celgene, Novartis: Research Funding. Mikhael:Onyx: Research Funding; Celgene: Research Funding; Sanofi: Research Funding; Novartis: Research Funding. Stewart:Celgene Novartis Array BioPharma BMS, research funding Millenium: Consultancy, Research Funding.

Abstract Background:A goal of “post-genomic” cancer care is development of specific, affordable, reliable and rapid next-generation sequencing-based assays determining prognosis, therapeutic selection and minimal disease monitoring, replacing older technologies in the diagnostic and monitoring workup. In this regard, we designed and deployed a custom gene panel for identification of mutations, copy-number changes and clonal frequency in multiple myeloma (MM) samples. Methods: The MM Mutation Panel Version 2.0 (M3P 2.0)1 includes 1271 amplicons from 77 genes that are either mutated in MM (genes with a published mutation incidence ≥3%), represent critical pathophysiological pathways, are targetable with small molecule-based therapies and/or are associated with drug resistance (eg. XBP-1, CRBN, IZKF, IRF4). The amplicons are spread across 21 chromosomes, allowing the identification of copy-number changes in these regions. Results: We have sequenced 38 MM cases (150 will be presented) with paired tumor-germline DNA samples, using only 20ng of DNA for library preparation. Patients (Pts) included 29 untreated and 9 MM pts with multi drug refractory disease. Coverage depth averaged 964X. Hyperdiploid MM was successfully identified in 45% of cases, del(13q) in 34%, del(1p) in 18%, del(17p) in 8% and gain of 1q in 13%. Array CGH in representative cases confirmed concordance with semiconductor sequencing. Overall, 71 exonic missense and 3 nonsense mutations were detected, and 7 frameshift indels. In 89% of the pts a mutation in at least one M3P2.0 gene was identified (range 1-8). 92% of mutations were predicted damaging (Provean, Polyphene-2 or SIFT). Most commonly mutated were KRAS (37%), NRAS (21%), BRAF (13%), TP53, CDKN1B, DIS3 (each 11%), FAM46C, STAT3, and IRF4 (each 8%). The MEK-ERK pathway was mutated in 61% of cases and in 3 cases more than one gene in this pathway (NRAS, KRAS, BRAF) was simultaneously mutated. Six mutations in BRAF were identified in 5 untreated pts, 3 of them known to be activating and targetable (Val600E). Other commonly mutated pathways were the NFKB pathway in 24% of pts and the Cyclin D pathway, mutated in 16% of cases. Genes of the cereblon pathway (CRBN, IRF4, IKZF3), essential for the action of lenalidomide (len) were mutated in 5 Pts. One newly diagnosed and len refractory Pt harbored a subclonal CRBN mutation (Asn316Lys, allelic fraction [AF] 20%) as well as 4 clonal IRF4 mutations within the DNA binding domain of the gene (Lys89Asn (AF 78%), Lys77Glu, Ser48Arg and Cys49Ser (AF 80% each). Another IRF4 mutated (Lys123Arg, AF 90%) hemodialysis dependent Pt was responsive to dose adapted treatment of len and dexamethasone (dex). Mutations in the C2H2-type 3 region of IKZF3 in a len resistant (Gly191Arg, AF 48%) pt occured as well as in a len and dex responsive (Arg242Gly, AF 52%) Pt. Furthermore, we identified a truncating XBP1 mutation (p.Leu232*, 97% AF) in a Pt refractory to bortezomib. One patient harbored a mutation in the steroid receptor NR3C1 (Pro255Leu, 22% variant reads). Finally, integrated mutation and copy-number data identified biallelic deletions of CDKN2C and FAM46C in a multi drug refractory Pt. Mutation and/or copy-number losses in both alleles were found in DIS3 in 4 Pts, in CYLD, TP53 and TRAF3 in 2 Pts and in CARD11, CDKN2A, CDKN1B, RB1and STAT3 in one Pt each. Conclusion: In summary, we report the initial results from 38 MM Pts using a custom MM sequencing panel which employs small amounts of DNA requiring very few cells and providing deep coverage in clinically meaningful timeframes. Our findings frequently include prognostically significant information, actionable targets and mutations in genes related to drug resistance. Targeted mutation profiling will likely become part of the clinical workup in MM and our M3P2.0 mutation panel is a suitable tool to provide information needed to guide precision therapy and to set the basis for individually tailored treatment decisions. 1. Kortuem KM. et al. Development Of a 47 Gene Sequencing Panel For Common Mutations Present In Multiple Myeloma: Preliminary Results In 77 p53 Deleted Patients, ASH 2013. Disclosures Stewart: Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Array BioPharma: Consultancy; Sanofi: Consultancy; Takeda Pharmaceuticals International Co.: Research Funding; Novartis: Consultancy. Bruins:OncoSpire: Salary support Other. Vasmatzis:OncoSpire: Salary Support Other. Kumar:Celgene: Consultancy, Research Funding; Millennium: The Takeda Oncology Co.: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding; Sanofi-Aventis: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Skyline Diagnostics: Membership on an entity's Board of Directors or advisory committees. Fonseca:Medtronic, Otsuka, Celgene, Genzyme, BMS, Lilly, Onyx, Binding Site, Millennium, AMGEN: Consultancy, patent for the prognostication of MM based on genetic categorization of the disease. He also has sponsored research from Cylene and Onyx Other, Research Funding. Bergsagel:MundiPharma: Research Funding; OncoEthix: Research Funding; Constellation Pharmaceutical: Research Funding; Novartis: Research Funding.

Abstract Background: It is now well established that the immune modulator (IMiD) drugs thalidomide (Thal), lenalidomide (Len) and pomalidomide (Pom) bind a specific pocket in the protein, cereblon (CRBN) that is required for MM cytotoxicity after IMiD treatment. Recently, we reported 46 CRBN binding proteins which were altered by Len, two of which were the transcription factors Ikaros (IKZF1) and Aiolis (IKZF3), important regulators of B and T cell development. Others had previously reported the same finding and went further by demonstrating that following binding of IMiD drugs, CRBN accumulates and accentuates the ubiquitination and subsequent proteasomal degradation of these two binding proteins. The resulting degradation and depletion of IKZF proteins has the simultaneous effect of downregulating IRF4 and MYC leading to MM cell death while simultaneously enhancing transcription of interleukin 2 and activating the immune compartment. Methods: To further explore this phenomenon and its relationship to drug sensitivity we first created two stable MM cell lines (H929IKZF1Luc, 8226IKZF1Luc) expressing an IKZF1-luciferase (luc) fusion gene and tested for sensitivity to Len alone and in combination with dexamethasone (Dex), bortezomib (Bor) or the histone deacetylase inhibitor SAHA. Next, we constructed an adenoviral vector expressing IKZF1-luc and used this to transfect 16 MM cell lines, confirmed expression and tested sensitivity to Len by MTT (Figure 1). Results: We first examined the stable cell lines and measured luc expression after exposure to all three IMiD drugs. With all IMiD drugs luc dramatically decreased in sensitive H929 cells. IKZF1 depletion was both time and dose dependent. Using this surrogate marker cell line assay, luc is most efficiently degraded by Pom which is twice as potent as Len, but a thousand times more potent than Thal (Concentration of drug required to reduce luc expression by 50%, Pom: 4.9nM, Len: 10.2 nM, and Thal: 4,795 nM). We then tested all 16 cell lines with Len 2uM for 5 hours and show a strong correlation of IKZF-luc depletion with sensitivity. Figure 1: Luc levels 5 hours after Len treatment at 2uM. We divided the cell lines into three groups: 1) Len resistant: 8226, KMS12PE, My5, SKMM2, JJN3, FR4, H112; 2) Len intermediate response: EJM, OPM2, U266, KMS18; 3) Len sensitive: H929, MM1.S, MM1.R, KMS11, XG1; Figure 1:. Luc levels 5 hours after Len treatment at 2uM. We divided the cell lines into three groups: 1) Len resistant: 8226, KMS12PE, My5, SKMM2, JJN3, FR4, H112; 2) Len intermediate response: EJM, OPM2, U266, KMS18; 3) Len sensitive: H929, MM1.S, MM1.R, KMS11, XG1; We next performed western blots on 9 MM cell lines and demonstrate that IKZF1 levels were lowest in 3 of 5 resistant MM cell lines and normal in all 4 sensitive lines studied. The cell line (FR4) with the lowest levels of IKZF1 harbors a damaging mutation of IZKF1 as well as a translocation which upregulates IRF4, an IKZF target. Of the remaining two resistant lines one has low levels of Cereblon. In one line the mechanism of IMiD resistance could not be established. Finally, we were curious about the dichotomy of the IMiD biology which seemingly has an absolute requirement for a functional proteasome for successful IMiD cytotoxicity which is at variance with clinical reports of synergy between the IMiDs and the proteasome inhibitors (PIs) Bor and carfilzomib (Car). To explore this, we treated the 8226IKZF1Luc cell line with Len alone or in combination with increasing concentrations of Bor (6 nM) or Car (40nM) and found that both totally blocked IKZF1 fusion protein degradation promoted by Len, beginning as soon as 1 hour at 100nM Bor or after 3 hours at 40nM treatment. Conversely, in low concentration of Bor (<4nM) no IKZF1 degradation blocking was observed in the Luc assay and Bor remained lethal to the MM cells. In that light with lower levels of Bor (<3nM) even varying the sequencing of MM cell exposure to the two drug classes did not block cytotoxicity. Other MM active drug Dex or SAHA, blocking the aggresome, had no significant inhibition for Len-induced IKZF1 fusion protein degradation. Conclusion: These results validate the central role for the CRBN-IKZF-IRF4 axis proteins in IMiD response and show a strong correlation of IZKF1 expression and the ability to degrade IKZF1 with sensitivity. Our data provide interesting and perhaps disconcerting biologic data suggesting that the timing and dosing of clinical use of IMiDs and PI treatment in combination should be carefully evaluated to prevent antagonism of effect but suggest in vitro that a therapeutic window exists at which proteasome inhibitors can be active without blocking IMiD activity. Disclosures Stewart: Sanofi: Consultancy; Array BioPharma: Consultancy; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Takeda Pharmaceuticals International Co.: Research Funding.