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Abstract Background: The clinical impact of ibrutinib is irrefutable and has been hailed as a revolutionary treatment for several cancers, including mantle cell lymphoma (MCL). Despite high response rates noted with ibrutinib therapy in MCL (ORR, 68%), complete responses are low and durability of remission is suboptimal in MCL vs. CLL or Waldenstrom macroglobulinemia (WM) patients (Cheah et al Ann Oncol 2015). This relative lack of efficacy may be due to MCL cells having a higher proliferation rate and being less indolent in nature than CLL or WM cells. Tumor cells with a high proliferation index rely on optimally functioning protein metabolism/homeostasis systems. In MCL, this observation has been fortuitously exploited with the use of bortezomib, a 20S proteasome inhibitor. Indeed the ubiquitin proteasome system is a validated target and consists of several individual components whose function in MCL has yet to be mapped out. We have recently reported that inhibition of the 19S proteasomal deubiquitinating enzymes (DUBs), USP14 and UCHL5; with the small molecule inhibitor b-AP15 is lethal to drug-resistant WM tumor cells. This intriguing observation, prompted us to define the role of USP14/UCHL5 and their inhibition in a diverse panel of B-cell lymphoma cell lines, including MCL models as well as ibrutinib-resistant MCL cells. Materials: b-AP15 (and its clinical grade analog, VLX1570) was obtained from Vivolux AB and bortezomib from Sellekchem. Human NHL (DLBCL, MCL, Burkitt), Hodgkin lymphoma (HL), multiple myeloma (MM) cell lines as well as established drug-resistant subclones were used in drug-sensitivity screening experiments (total n=25). Results: USP14 and UCHL5 mRNA expression was queried in the CCLE database (Broad-Novartis) and showed highest expression in plasma cell and B-lymphoid tumor cell types. This was also observed at the protein level, wherein analysis of The Human Protein Atlas database revealed moderate to high intensity staining for USP14 and UCHL5 localizing to the cytoplasmic/membranous and nuclear compartments in high-grade NHL tissues. Next, we examined B-lymphoid tumor cell sensitivity to pharmacologic inhibition of USP14/UCHL5 with either b-AP15 or VLX1570 (72hr fluorescein diacetate hydrolysis assay or CellTiter Glo). We observed that MCL cells, including those resistant to ibrutinib (Jeko/IR cell line) were highly sensitive to VLX1570 (median IC50 12 - 25nM). In contrast, MM cells were not as sensitive (IC50's ranging between 58 - 90nM). Nine DLBCL cell lines exposed to equivalent time/concentrations of b-AP15 showed a median IC50 of 311nM (range, 70 - 910nM) and 3 HL models exhibited an IC50 of 531nM (range, 420 - 783nM). No difference in sensitivity to VLX1570 was noted between ABC-type vs. GCB-type DLBCL models. These functional results aligned with prior genomic inquiry where USP14 and UCHL5 gene expression was noted to be higher in MCL cell lines compared to DLBCL and non-hematologic cancer models analyzed (FDR=0.0019, p=0.001), suggesting their more central role in maintaining MCL tumor viability. Indeed MCL tumor survival (particularly ibrutinib-resistant cells) appears linked to USP14/UCHL5 function as treatment of Jeko/IR cells with VLX1570 (250nM, 12hrs) caused marked apoptosis (71.8% annexin-V+ staining) and cleavage of PARP-1. Moreover, the combination of ibrutinib and VLX1570 synergistically induced MCL cell death (median CI: 0.3). Mechanistic studies demonstrated that VLX1570 modulates several proteins critical for MCL survival including cyclin-D1, Myc and CXCR4/CD184. Reduction in cyclin-D1 was also noted to coincide with cell cycle arrest of VLX1570-treated MCL cells. Altogether, these findings suggest that VLX1570 modulates critical cellular signaling pathways utilized by MCL tumor cells, particularly those that are involved in resistance to ibrutinib. Conclusions: MCL remains a challenging malignancy to treat with no effective treatments for ibrutinib-relapsed disease. Our data supports the role of the proteasome-associated DUBs, USP14 and UCHL5, as critical factors involved in ibrutinib-resistant MCL tumor survival. VLX1570 is a potentially effective agent for the treatment of ibrutinib-resistant MCL. Disclosures Gullbo: Vivolux AB: Other: Shareholder. Linder:Vivolux AB: Other: Shareholder.

Abstract Background In the U.S. over 14,000 new cancer cases occur annually in people under 21 years of age; approximately 21% of cases are acute lymphoblastic leukemia (ALL) and ∼ 30% classify as Burkitt lymphoma (BL). Most cases of ALL and BL are due to malignant B-cells that have acquired disease-defining oncogenic lesions that facilitate their rapid proliferation. To maintain a state of homeostasis, the malignant cells rely heavily on the ability of the internal degradation and recycling systems to match the high rate of protein turnover. As such, the ubiquitin proteasome system (UPS) is central in this operation where it prevents buildup of misfolded proteins while also regulating the availability of anti-apoptotic proteins such as BCL2 and MCL1. These observations coupled with the observation that UPS disruption leads to cellular demise have resulted in the development of several proteasome inhibitors (PI). These PI have primarily been examined in adult B-cell malignancies. The feasibility of a bortezomib-containing regimen for use in pediatric malignancies has been examined by the TACL consortium and demonstrated a 73% response rate in relapsed/refractory ALL patients. However, a major limitation of bortezomib-based therapy is an increased incidence of peripheral neuropathy (PN), which was also noted in the TACL study. As such, more potent and reportedly less toxic PI have been developed that seem to carry considerably lower risk for development of PN. Such agents that maintain a high response rate and carry reduced long-term adverse effects are highly desirable in pediatric age patients. Aim Investigation of the novel proteasome inhibitors carfilzomib and MLN9708 (Millennium Pharmaceuticals) in preclinical models of pediatric B-lymphocytic cancer. Methods Representative preclinical models of BL (Raji cell line) and B-ALL (ALL-1 cells) were used in this study. Proteasomal activity was measured using synthetic fluorogenic peptide substrates. Cell viability was measured by MTS assay. Apoptosis was determined by annexin-v/PI staining and mitochondrial membrane permeability (MOMP) was assessed using TMRM followed by flow cytometry. Protein profiling was performed via immunoblot. Results In an effort to understand the effect of carfilzomib and MLN9708 in our pediatric cancer models, we first assessed their ability to inhibit proteasomal activity. While chymotrypsin-like activity, which is the target of both PIs, is inhibited by more than 90% in both cell lines, inhibition (∼ 35%) of caspase-like function conferred by the b1 subunit was more significantly pronounced in MLN9708 treated Raji and ALL-1 cells. Next we performed an MTS assay to determine the growth inhibitory effects of these PI in vitro. Both PI inhibited the growth of the Raji and ALL-1 cells in a dose dependent manner. Carfilzomib showed a more potent effect (IC50 ∼1nM in both cell lines) as compared to MLN9708 (IC50 ∼30nM in ALL-1 cells and ∼50nM in Raji cells). To further understand mechanisms of growth inhibition we examined both tumor cell lines for induction of apoptosis along with corresponding markers. As compared to Raji cells, we observed ALL-1 cells to be more sensitive to the effects of both PI, albeit with MLN9708 (50nM) inducing more cell death (65%) as compared to carfilzomib (10nM, 50% cell death). Further, we observed altered MOMP, cleavage of caspases 9, 3 and PARP-1 were found to occur in both cell lines in presence of carfilzomib or MLN9708. These changes indicate the apoptotic effects of novel PI in ALL-1 and Raji cells are via mitochondrial destabilization and caspase activation. As inhibition of the UPS aids NOXA and BIM (pro-apoptotic BCL2 proteins) mediated cell death, we examined the expression of BCL2 family proteins changes in Raji and ALL-1 cells in response to novel PI. Indeed protein levels of both BCL2 and MCL1 were reduced in carfilzomib and MLN9708 treated cells. Reduced BCL2 expression was more pronounced in ALL-1 cells as compared to Raji cells and downregulation was noted to be dose-dependent. Summary Our data show that both carfilzomib and MLN9708 potently inhibit the viability of malignant ALL and BL cells in vitro. We conclude that PI are promising anti-neoplastic therapeutics that target not only the UPS but also modulate the expression of critical anti-apoptotic proteins. Data from mechanistic studies conducted herein underscore the need for further research in in vivo models of these diseases. Disclosures: Foran: Celgene: Research Funding.

Abstract Background: Dexamethasone (dex) is an integral part of various anti-myeloma regimens both in the frontline and relapsed/refractory (RR) setting. Several clinical trials have shown that dex adds to the efficacy of novel therapeutic agents for multiple myeloma (MM). Yet, a substantial number of patients are intolerant to even the standard of care low-dose dex (40 mg weekly), especially in heavily pre-treated RRMM, possibly due to cumulative steroid toxicities and thus, require dex dose adjustments. There is a concern of compromising the efficacy of anti-MM regimens by adjusting the dex dose lower. We describe the frequency of dex dose adjustment, characteristics of patients who required it and its effect on patient outcomes in a large prospective clinical trial of pomalidomide (pom) and dex in RRMM (MC0789). Methods: We utilized data from the Pom-dex trial (MC0789), which treated RRMM patients in different cohorts of pom (2 vs. 4 mg; 21 vs 28 days cycle) with fixed dose dex (40 mg orally weekly). Patients were divided into 2 subgroups, with any dose adjustment/elimination of dex vs. not. Continuous and categorical variables were compared between the two groups using two-sample t-test/Wilcoxon rank sum test or Chi-squared test, respectively. Logistic regression and Cox regression models were used to find the univariate and stepwise multivariate predictors of response to treatment and overall mortality, respectively. Kaplan-Meier method was used to estimate overall survival (OS) at 1, 3, and 5 years. All statistical tests were two-sided with the alpha level set at 0.05 for statistical significance. Results: Results from the MC0789 clinical trial have been reported previously. Almost half of all treated patients in this trial had dex dose adjusted (n=175/343; 51%). The median number of cycles for the first dose adjustment in this group was 3 (range 1-62), with 46.5% of patients (n=82) having multiple dose adjustments and majority (58.3%, n=102) within the first 3 cycles of treatment. Patients in the dose adjustment group were significantly older (mean 65 vs 60 years; p=0.0001). Males, Whites and patients diagnosed before 2006 or with normal cytogenetics also had a higher incidence of dex dose adjustment, though this was not statistically significant. Patients with dex dose adjustment had a longer duration of treatment (mean 339 days) as compared to the non-adjusted cohort (mean 123.65 days) (95% CI 160.13, 270.58; p <0.0001). Dex dose adjustment was associated with a higher probability of responding in the clinical trial after a univariate (p<0.001) as well as multivariate (p<0.001) analysis. Patients with dex dose adjustment also had a significantly better OS at 1, 3 and 5 years by univariate (p<0.001) and multivariate (p<0.001) analysis. (Figure 1) The only other significant factor was lesser number of prior regimens (3 or less), associated with a higher probability of response (p=0.002) and survival (p<0.001). Conclusion: Our analysis established that in these heavily pre-treated RRMM patients, appropriate dose adjustment of dex did not compromise their response rates; patients remained on treatment significantly longer and had an improved survival. Majority of patients (58.3%) requiring dex dose adjustment had it instituted early during treatment (<3 cycles), suggesting that early disease progression was less likely to determine dex dose continuation or adjustment. Appropriate dex adjustments should be judiciously employed as they may ensure longer treatment duration with novel anti-MM therapeutic agents, conferring superior response and survival in this difficult to treat MM subgroup. This also paves the path to further optimize dex dose in various anti-MM regimens, especially in these patients who are more likely to be intolerant to it. Disclosures Kumar: Skyline, Noxxon: Honoraria; Celgene, Millennium, Onyx, Janssen, Noxxon, Sanofi, BMS, Skyline: Consultancy; Celgene, Millennium, Onyx, Novartis, Janssen, Sanofi: Research Funding.

Abstract Dexamethasone is a powerful anti-inflammatory, immunosuppressive agent widely used in the treatment of MM with pleiotropic effects on both the tumor, and host immune cells. We report on a completely novel approach to immune modulation using an agent with almost directly opposite effects to glucocorticoids on innate immune cells. LCL161 is an IAP antagonist targeting the cellular inhibitor of apoptosis proteins cIAP1 and -2 (cIAP1/2) that in a phase I study in solid tumors was well tolerated with a cytokine release syndrome as the dose limiting toxicity. We previously reported that loss of cIAP1/2 in MM cells results in the stabilization of NIK and activation of the non-canonical NFkB pathway, a pathway we found frequently activated by a promiscuous array of mutations in multiple myeloma (MM). As expected, we found that LCL161 has no direct anti-tumor activity against MM cells in vitro and ex vivo. However, the non-canonical NFkB pathway is also a key activator of the innate immune response. Remarkably, we found that in vivo LCL161 has dramatic, apoptosis-independent activity associated with marked phagocytosisagainst MM that develops spontaneously in an immunocompetent genetically engineered mouse model of MM (Vk*MYC). Cyclophosphamide (Cy), which has been shown to induce an acute secretory activating phenotype in tumor-associated macrophages, markedly potentiated the anti-tumor effects of LCL161. We show that phagocytes (depleted by liposomal clodronate), but not adaptive immune cells, are required for the anti-tumor effect. Consistently we show that LCL161 activates dendritic cells and macrophages in vitro, and in vivo, and upregulates production of inflammatory cytokines. A phase 2 clinical trial of LCL161 was conducted in 24 patients with relapsed MM previously exposed to both IMiDs and PIs, but less than 5 prior lines of therapy. Patients received LCL161 1200mg po weekly, with Cy 500mg po weekly added for progressive disease or lack of response. Single agent LCL161 was well-tolerated, with grade 3 or 4 non-hematologic toxicity in four patients, and following the addition of cyclophosphamide in 9 patients. No patients responded to single agent LCL161, and the best response was stable disease in 6/24. In 21 patients, Cy was added after a median of 2 cycles, in whom the best response was stable disease or better in 18/21 (p<0.001 compared to LCL161 alone), and a PR or better (including 1 VGPR and 1 CR) in 5/21 (p<0.001 compared to LCL161 alone). Six months following the initiation of Cy, 35% of patients remain event-free. Two patients had stable disease for more than nine months after stopping treatment. One, relapsing after VDT-PACE, HDM200, RVd x 3 years with t(11;14) and del17p, received only three months of LCL161, the last month in combination with Cy. He then achieved a PR, reconstitution of his uninvolved IgG to normal levels for the first time in more than 3 years and still has not progressed one year after stopping therapy. Similar changes in serum and bone marrow inflammatory cytokines and gene expression were observed in patients following treatment with LCL161 to those observed in the mice. We conclude that LCL161 is able to modulate the tumor microenvironment to exert long-term disease control in combination with Cy, and deserves further study in combination with agents that more directly depend on phagocytic cell activity (e.g., monoclonal antibodies, inhibitors of "Don't eat me" signals). Disclosures Bergsagel: Novartis: Research Funding; Constellation Pharmaceuticals: Research Funding; Incyte: Consultancy; Jannsen: Consultancy; Mundipharma: Consultancy. Off Label Use: Use of LCL161 to treat multiple myeloma. Kumar:Celgene: Research Funding; Millenium/Takeda: Research Funding; Onyx: Research Funding; AbbVie: Research Funding; Janssen: Research Funding; Sanofi: Research Funding; Celgene, Millenium, Sanofi, Skyline, BMS, Onyx, Noxxon,: Other: Consultant, no compensation,; Skyline, Noxxon: Honoraria. Stewart:Oncospire Genomics: Research Funding.

Abstract Background: Lenalidomide (Len) is clinically active in CLL patients (pts). Robust anti-leukemic immune response from Len is truncated by dysfunctional immune system in CLL and anti-apoptotic bcl-2 protein. We hypothesized that therapeutic downregulation of Bcl-2 may help enhance the killing potential of immune effector cells that are activated by Len. AT-101 is a novel, orally active BH3-mimetic that binds to antiapoptotic Bcl-2 family proteins (Bcl-2, Mcl-1 and Bcl-xL) and induces CLL cell death ex vivo (Masood et al British Journal of Haematology 2012). Encouraging efficacy and safety results of AT-101 alone or in combination with rituximab in CLL have been reported in Phase I/II studies. We have previously demonstrated that bcl-2 downregulation in CLL cells enhanced the killing potential of Len-activated immune cells. Conversely, pretreatment of CLL cells with Len enhanced AT-101 cytotoxicity in an immune cell independent manner (Masood et al British Journal of Haematology 2012). These preclinical findings formed the basis of a phase I/II clinical trial testing the combination of AT-101 and lenalidomide in relapsed B-CLL patients. Methods: The phase I portion of this study (NCT 01003769) utilizes a standard cohort of three design to determine the maximum tolerated dose (MTD) of the combination regimen. The MTD is defined as the dose level below the lowest dose that induces dose-limiting toxicity (DLT) in at least one-third of patients. Following the MTD determination, the phase 2 portion will assess the efficacy of this combination using a one-stage design with an interim analysis. Key inclusion criteria include adult CLL pts with relapsed disease who have received 1-4 prior lines of treatment (Rx) (phase I) or 1-2 lines of Rx (phase II), last dose > 4 weeks from enrollment, ECOG performance status 0-2, good renal/liver function, acceptable blood counts. Patients with known hypersensitivity to thalidomide/Len or prior use of gossypol/AT-101 were excluded. Cycle 1 Rx includes Len alone and cycles 2-12 includes Len and AT-101. Starting doses of Len and AT-101 are 5mg oral daily on days 1-21 and 40 mg oral twice daily (b.i.d) on days 1-3 of a 28-day cycle. Planned dose escalation is shown in Table 1. Three pts will be treated at each dose level and observed during the first cycle of the combination Rx. Dose limiting toxicity is defined as grade 4 anemia unrelated to disease, thrombocytopenia-grade 4 or grade 3 with bleeding/requiring platelets, ANC <500 for >14 days, febrile neutropenia , Grade 3/4 non-hematologic toxicity. Dose from Phase II will be based on MTD from phase I. Peripheral blood and bone marrow will be collected and analyzed for immune cellular microenvironment and effect of Len and AT-101 on molecular targets. Optional lymph node biopsy will be done at baseline and if tumor-flare reaction occurs. This study is expected to accrue a maximum of 26 pts in phase I and 34 pts in phase II, overall maximum sample size of 60 pts. Five pts in phase I have been accrued to date. Table 1 (* starting dose level) Table 1. (* starting dose level) Disclosures No relevant conflicts of interest to declare.

Abstract Background: PRCA is characterized by severe normochromic normocytic anemia, reticulocytopenia, and markedly reduced bone marrow erythroid precursors with intact leukocytic and megakaryocytic lineages. There is limited information about the prevalence and outcomes of PRCA in CLL, with the vast majority of information derived from case reports and small series comprising <10 patients. Here, we describe the clinical features, therapy, and outcomes of CLL patients with PRCA seen at our institution. Methods: The Mayo Clinic CLL Database includes all patients with a diagnosis of CLL evaluated at Mayo Clinic Rochester, MN between January 1995 and December 2014. We identified patients diagnosed with PRCA per the following diagnostic criteria: those who presented with normocytic anemia, had decreased erythroid precursors on bone marrow examination, and reticulocytopenia that could not be explained by any other cause besides PRCA. The baseline demographics, clinical characteristics, therapy, and outcomes were abstracted from the electronic medical records. We generated descriptive statistics and calculated overall survival (OS) from diagnosis date (PRCA, autoimmune cytopenia [AID], or CLL) to last known alive or death date. OS was plotted using Kaplan Meier curves and compared between groups using log-rank statistic. The Mayo Clinic IRB approved this study. Results: Of the 3945 CLL patients seen during the study interval, we identified 30 patients who met the diagnostic criteria of PRCA as described in the Methods section. The baseline characteristics, etiology and therapy for PRCA are shown in Table 1. The median time to PRCA from CLL diagnosis was 4.5 years (range-0.1-14 years). Of the 13 patients with IGHV results available, 12 (92%) were IGHV unmutated. Twenty-one (70%) were male and their median age at PRCA diagnosis was 67 years (range 40-81 years). Parvovirus testing was positive in 6 of 24 (25%) patients who were tested; 3 of these patients were treated with IVIG, 2 were treated with prednisone and IVIg, and 1 was treated with prednisone and rituximab. Eight patients (27%) had both PRCA and autoimmune hemolytic anemia (AIHA), and 1 patient (3%) had concomitant PRCA and immune thrombocytopenia (ITP). Of the 27 patients for whom we have treatment data, 10 required only one treatment regimen and 17 required 2-6 treatment regimens, with two patients requiring splenectomy for concomitant AIHA. The median survival of CLL patients after a diagnosis of PRCA was 4.6 years (Figure 1). There was no difference in survival between PRCA cases with and without parvovirus (p=0.92). Compared to newly diagnosed CLL Rai stage III/IV patients, there was no difference in OS (p=0.53). There was no statistically significant difference in survival from an AID cytopenia diagnosis when comparing PRCA, ITP, AIHA, and Evans syndrome (Figure 1; median 5 years for PRCA and 6 years for other groups; p=0.17). Conclusion: PRCA is a rare complication in CLL, occurring in <1% CLL patients seen at our institution over the past 20 years. Parvovirus was the etiologic factor in ~25% of patients and did not influence outcomes. There appears to be no significant difference in outcomes of PRCA patients compared to patients with other AID in CLL. Table 1. Patient Characteristics Characteristic N [range] or (%) Number of patients 30 Males 21 (70) IGHV status* · Unmutated · Mutated 12 (92) 1 (8) FISH results at CLL diagnosis* · 17p- · 11q- · Trisomy 12 · 13q- · Negative 0 (0) 1 (10) 3 (30) 3 (30) 3 (30) At PRCA Diagnosis Median age 67 [40 - 81] Median hemoglobin (gm/dL)* [normal range 12-15.5 gm/dL] 8.2 [3.5 - 10.8] Median absolute reticulocyte count (x 109/L)* [normal range: 38 - 113 x 109/L] 3.5 [0.0 - 25.3] Patients treated for CLL prior to PRCA diagnosis 25 (83) Etiology of PRCA Parvovirus (n = 24) 6 (25) Therapy-related: · Fludarabine · Alemtuzumab/rituximab · Pentostatin/cyclophosphamide/rituximab 3 (10) 1 (3) 1 (3) Autoimmune (neither therapy nor parvovirus related) 19 (63) Initial Therapy of Non-Parvovirus Related PRCA Steroids alone 2 (8) Steroids and Rituximab 3 (13) Steroids in combination with: · IVIG, Chlorambucil · Cyclosporine · Rituximab, IVIg · IVIg, splenectomy 1 (4) 2 (8) 1 (4) 1 (4) Monoclonal Ab alone · Alemtuzumab · Rituximab 1 (4) 4 (17) Cyclosporine 1 (4) Cyclophosphamide 2 (8) RCP (Rituxan, Cyclophosphamide, and prednisone) 2 (8) IVIg 1 (4) Not available 3 (13) *Not available for all patients Figure 1. Figure 1. Disclosures Ding: Merck: Research Funding. Kay:Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Tolero Pharma: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding. Shanafelt:Cephalon: Research Funding; Hospira: Research Funding; Glaxo-Smith-Kline: Research Funding; Genentech: Research Funding; Jannsen: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding; Pharmacyclics: Research Funding.

Abstract Background Severe cytopenias in CLL are typically associated with bone marrow failure due to massive infiltration of the bone marrow by leukemic cells and negatively impact patient morbidity/mortality. Although near complete marrow replacement by CLL is typically observed in patients whose degree of cytopenia meet criteria for advanced Rai stage disease (Hg<11g/dL; platelet <100 x10^9/L), profound variability in the degree of cytopenia and the extent of marrow infiltration exists, and the relationship between these dimensions is not well characterized. Methods We used the Mayo Clinic CLL database to identify all previously untreated CLL/SLL patients who underwent bone marrow biopsies. CLL patients known to have autoimmune-based cytopenias were excluded from this study. Hemoglobin, platelets, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), and absolute monocyte (AMC) at the time of bone marrow biopsy were collected. Characteristics from bone marrow biopsy including marrow cellularity, % CLL involvement, CLL leukemic index (calculated as % cellularity times % CLL), and non-CLL leukemic index (calculated as % cellularity times (1-% CLL)) were also collected. Bone marrow cellularity and % CLL involvement were recorded in the Mayo Clinic CLL database based on clinical estimates made by Mayo hematopathologists. Clinical characteristics and CLL prognostic factors (e.g., Rai stage, CLL FISH, IGHV, ZAP-70, CD38 and CD49d) were also obtained from the database. Age- and sex-adjusted Pearson correlations between the bone marrow measurements and hemoglobin and platelet levels were computed, and scatterplots were created. Results A total of 695 untreated CLL patients diagnosed between 1972 and 2015 who had a CBC test at the time of bone marrow biopsy performed at Mayo Clinic were identified. Median age at diagnosis was 61 years, and 487 (70%) patients were men. Mean time from CLL diagnosis to bone marrow biopsy was 2.2 years. At the time bone marrow biopsy was performed, 20% of patients had advanced stage disease (Rai III/IV) disease, while 44% and 36% had intermediate (Rai I/II) and early stage (Rai 0), respectively. With respect to prognostic parameters, 53% had unmutated IGHV and 20% had high risk CLL FISH (11q- and 17p-). The distribution of other prognostic factors included 43% CD49d+, 35% CD38+, and 42% ZAP-70+ CLL diseases. Median peripheral blood counts were: hemoglobin 13g/dL, platelet 167,000/mL, ALC 26,600/mL, ANC 4100/mL, AMC 700/mL. The median bone marrow cellularity and percent CLL involvement were 70% and 70%, respectively. The median CLL leukemic index was 42%, and median non-CLL leukemic index was 21%. Pearson correlation analysis of bone marrow measurements with peripheral blood hemoglobin found the strongest correlation with the CLL leukemic index (r= -0.49, p<0.001) (Figure), followed by the % marrow CLL involvement (r= -0.45, p<0.001), cellularity (r= -0.40, p<0.001), and non-CLL leukemic index (r= 0.37, p<0.001). Pearson correlation analysis of the bone marrow measurements with platelet count also found the strongest correlation with the CLL leukemic index (r= -0.39, p<0.001), followed by % marrow CLL involvement (r= -0.38, p<0.001), non-CLL leukemic index (r= 0.35, p<0.001), and cellularity (r= -0.28, p<0.001). The peripheral blood ANC levels did not correlate with the four bone marrow measurements. Age- and sex-adjusted Pearson correlations between the bone marrow measurements and hemoglobin and platelet levels were similar to the above unadjusted results. Conclusion To our knowledge, this is the first study to examine the relationship between peripheral blood counts and the extent of bone marrow infiltration in patients with CLL. Although the bone marrow leukemic index had the strongest correlation with peripheral blood Hg and platelet counts, the correlation was modest with an r value of -0.49. These findings suggest that the extent of infiltration of the bone marrow by CLL cells only partially explains of the anemia and thrombocytopenia observed in CLL, indicating other biologic interactions between CLL B-cells and hematopoietic cells contribute to anemia and thrombocytopenia in these patients. More research is needed to determine the nature of these interactions. Disclosures Ding: Merek: Research Funding. Kay:Genentech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharm: Research Funding. Shanafelt:genentech: Research Funding; Glaxo-Smith-knine: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding; Hospira: Research Funding; Polyphenon E Int'l: Research Funding.

Abstract Background Waldenströms macroglobulinemia (WM) is a lymphoplasmacytic subtype of Non-Hodgkins lymphoma in which specific molecular changes lead to unrestricted cell proliferation and overproduction of immunoglobulin-M. These processes require exquisite coordination between growth promoting transcription factors (NFkB) and cell homeostatic systems. The bioavailability of various transcription factors and cell cycle proteins is primarily regulated by the ubiquitin proteasome system (UPS). Moreover, the proteolytic machinery ensures proper elimination of unwanted or misfolded proteins, which when disrupted leads to protein accumulation and apoptotic cell death. The 26S proteasome is a barrel shaped structure with a 20S catalytic core that is flanked by 19S caps on either side. 26S proteasomes are critical for WM cell survival, which is clinically evident, as up to 80% of WM patients treated with the proteasome inhibitor (PI) bortezomib have derived significant benefit. Traditional PI such as bortezomib and carfilzomib have been designed to target the B5 catalytic core within the 26S proteasome. However, proteasomes are capable of altering conformation of their proteolytic subunits in response to inflammation and stress, which can lower the effectiveness of these PI. The 19S cap is the regulatory complex and functions in unfolding and deubiquitinating the proteins before their entry into the 20S complex using constitutive deubiquitinating enzymes (DUBS). Recently, it has been demonstrated that inhibition of the DUBS, UCHL5 and USP14 results in cell death in a variety of tumor types but more so in cancer cells that rapidly divide or have a high protein turnover rate. Aim To define the anti-WM activity of the novel DUB inhibitor, b-AP15 in preclinical models of WM. Methods The WM cell lines BCWM.1, RPCI-WM1 and MWCL-1 were used for experiments in this analysis. Proteasomal activity was measured using synthetic fluorogenic peptide substrates. Apoptosis was determined by annexin-V/PI staining and mitochondrial membrane permeability (MOMP) was assessed using TMRM followed by flow cytometry. Protein profiling was done via immunoblot analysis. Results As reported by D’Arcy et al in their characterization of the b-AP15 molecule, inhibition of UCHL5 and USP14 by b-AP15 does not disrupt proteasomal activity conferred by the b-catalytic subunits. We confirmed these findings by treating WM cells with single agent bortezomib (10nM), carfilzomib (10nM), b-AP15 (100nM) or all three agents in combination to assess chymotrypsin-like, caspase-like and trypsin-like proteasomal activity using fluorogenic peptide substrates. Further, when used in combination with the other PI, PI-mediated chymotrypsin-like activity remained inhibited in the presence of b-AP15. Next, we sought to determine if b-AP15 treatment could induce WM cell death. Annexin-v staining showed that b-AP15 (500uM) induced apoptosis in all WM cell lines (28 – 60% cell death) with variable responses noted among the 3 models. Treatment with b-AP15 increased MOMP while also inducing the cleavage of caspases-9 and 3 and PARP-1 suggesting that the anti-WM activity of b-AP15 is mediated through the intrinsic apoptotic pathway. As components of the NFkB signaling cascade lie downstream to the UPS, we examined the effects of b-AP15 on this pathway. When treated with the DUB-inhibitor, nuclear translocation of NFkB was reduced in a time dependent manner. Interestingly, treatment with b-AP15 increased the phosphorylation of the mitogen activated protein kinase-p38 (p38) in all the WM cell lines tested. Treatment of WM cells with b-AP15 in presence of the p38 inhibitor, SB203580, resulted in a synergistic induction of cell death. Conclusion b-AP15 is a novel DUB inhibitor that disrupts two regulatory enzymes (UCHL5 and USP14) present in the 19S proteasomal cap. Results presented here demonstrate that b-AP15 induces apoptosis in in vitro models of WM and with in vivo analysis underway, is a potential therapeutic in WM. We would like to acknowledge the Waterfall Waldenström Macroglobulinemia Research Fund, the Leukemia and Lymphoma Society and the International Waldenström Macroglobulinemia Foundation for their continued support. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2861 Poster Board II-837 Bcl-2 protein family has the unique capability to balance between the cell survival and death by regulating the expression of its individual members. AT-101 is a BH3 mimetic and a potent inducer of noxa and puma, the natural ligands of BH3 family proteins. It is known to bind and inhibit the anti-apoptotic functions of Bcl-2 family members Bcl-2 and Bcl-XL and Mcl-1. In vitro it has been shown to induce apoptosis in several tumor models systems including multiple myeloma. In this report we investigated the effect of AT-101 on a model cell line, BCWM.1 (a known WM cell line, BCWM.1/WT), representing Waldenström Macroglobulinemia. This disease is characterized by the presence of lymphoplasmacytic cells in the bone marrow and the secretion of IgM monoclonal protein into the serum. Several conventional therapies are available but the disease remains incurable. Therefore there remains a need to develop new therapies for this orphan disease. Recently, bortezomib (a proteasomal inhibitor) has shown promising anti-WM activity with enhanced responses when combined with traditional therapies. But continued treatment with bortezomib result in the development of resistance in the clinic. We developed an in vitro model of bortezomib resistance from BCWM.1 (hereafter referred as BCWM.1/BR). These cells also developed cross resistance to conventional therapies used for WM such as fludarabine and doxorubicin. Biological characterization of this cell line demonstrated Bcl-2 as a potentially important therapeutic target. We therefore assessed the effect of AT-101 to identify preclinically if this could be a potential clinical strategy in future. AT-101 induced a dose and time dependent inhibition in the viability of both BCWM.1/WT as well as BCWM.1/BR cells. Cell death was observed at as low as 1uM concentration of AT-101 and at 10uM a maximum of 50-70% death was observed by 24h. While BCWM.1/WT cells showed a significant death at 12h, treatment with AT-101 induced cell death in BCWM.1/BR cells as early as 6h. These results indicate that AT-101 induced a potent and quick inhibition in viability in BCWM.1/BR cells as compared to their parental wild type cells. Investigation into the mechanism of cell death showed that AT-101 induced apoptosis in a mitochondrial dependent pathway in these cells. A comparative analysis of the signal transduction pathways operated in BCWM.1/WT and BCWM.1/BR cells showed that many of the cellular activation and survival pathways such as AKT, ERK1/2 that are present in BCWM.1 cells are inhibited in the resistant cells. Interestingly, BCWM.1/BR cells expressed a fivefold increase in the Bcl-2 protein as compared to BCWM.1/WT cells suggesting a Bcl-2 dependent survival of these cells in the absence of other cellular activation and survival signals. Increased susceptibility of BCWM.1/BR cells to AT-101 thus can be understood to be a direct consequence of an increased expression of Bcl-2 and a dependence of the resistant cells on Bcl-2 family of anti-apoptotic proteins for their survival. Results presented in this report suggest that AT-101 has a unique therapeutic potential against Waldenström Macroglobulinemia that is independent of resistance to bortezomib. These observations highlight bcl-2 as a potential target, and AT-101 as possible therapeutic avenue for WM patients. Disclosures: Chanan-Khan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immunogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract Background: The observation that B-cell receptor (BCR) signaling play a critical role in the survival of malignant Waldenstrom macroglobulinemia (WM) cells is highlighted by clinical efficacy of the BTK-inhibitor, ibrutinib in WM patients. BCR signaling leads to downstream activation of several intermediary components whose activity results in increased cell proliferation. Although disruption of this axis at the proximal end (BCR) has proved successful in WM, the exact role and impact of targeting the distal intermediaries remains unknown, particularly in aggressive forms of the disease. This is important as induction of resistance to ibrutinib is clearly documented which can either be mediated through mutational changes in BTK or other members within the BCR signalosome. Using a novel simulation-based approach, we reverse-engineered the WM cell molecular architecture to uncover the role of oncogenic intermediaries distal to the BCR complex in advanced-stage WM and conducted a virtual drug-screen targeting these pathways with in vitro validation. Methods: The human WM cell line, RPCI-WM1, was used in simulation and validation experiments. RPCI-WM1 was established from a highly drug-refractory advanced disease stage patient refractory to both rituximab and bortezomib. Publically available as well as proprietary genomic and cytogenetic data was utilized for the creation of the RPCI-WM1 patient avatar, which through simulation identified the salient and prominently dysregulated cellular pathways. Importantly, illustrating these pathways highlights common convergence points on increased proliferation and viability. These convergence points were then directly and indirectly targeted by simulated testing of a library of FDA approved drugs and those impacting these dysregulated pathways were shortlisted. A standardized library of equations models all the biological reactions such as enzymatic reactions, allosteric binding and protein modulation by phosphorylation, de-phosphorylation, ubiquitination, acetylation, prenylation and others. A library of FDA approved drug agents (n~150) and those in clinical study has been developed and was simulated individually and in combination on the RPCI-WM1 (advanced stage WM patient) avatar. Results: MYD88L265P mutation, absence of CXCR4 mutations and additional chromosomal aberrations (derivatives, translocations, deletions and amplifications of chromosomes 3, 6, 9, 13, 18 and 19), which are notable features of RPCI-WM1 cells were configured and modeled in silico. The RPCI-WM1 patient avatar was predicted to have increased IRAK1/4 engagement due to MYD88 mutation and high copy number (CN) of IL18. Downregulation of DUSP1 through MYD88-mediated signaling was noted to result in high ERK activity. Increase gene copy number of both FOS and ETS1, which are downstream of ERK, were noted. Notably, FOS is a key regulator of the AP1 complex, whose activity is regulated upstream by ERK. The transcription factor, ETS1 was also predicted as over expressed and ERK-mediated phosphorylation of ETS1 regulates the activity of ETS1. The RPCI-WM1 patient avatar model also indicated high AKT due to indirect convergence of multiple aberrations. Drug screening revealed sensitivity to the MEK inhibitor, binimetinib. Although no copy number variation in MEK-ERK pathway genes were detected, per simulation, MYD88 mutation through inactivation of DUSP1 activated ERK and downstream associated survival pathways. The simulation predictions were experimentally validated. As predicted, binimetinib significantly inhibited proliferation and viability of RPCI-WM1 cells (IC50 <100nM). Conclusions: Our study demonstrates the functional role and impact of MEK1/2 - an oncogenic component distal to the BCR, and whose activity can be targeted with binimetinib to elicit a lethal effect in advanced-stage WM cells. We also show the utility of a novel technology, which is capable of integrating genome-wide molecular data points to emulate the salient genomic drivers of a given tumor cell and test its sensitivity to numerous drugs in a high-throughput manner for truly personalized therapy. Disclosures Vali: Cellworks Group, Inc.: Employment, Equity Ownership. Kumar:Cellworks Group, Inc.: Employment. Singh:Cellworks Group, Inc.: Employment. Usmani:3Cellworks Research India Limited: Employment. Grover:3Cellworks Research India Limited: Employment. Abbasi:Cellworks Group, Inc.: Employment, Equity Ownership.