Citations

PurposeTo develop a robust, accurate, and accelerated T1ρ quantification solution for submillimeter in vivo whole‐brain imaging.MethodsA multislice T1ρ mapping solution (MS‐T1ρ) was developed based on a two‐acquisition scheme using turbo spin echo with RF cycling to allow for whole‐brain coverage with 0.8‐mm in‐plane resolution. A compressed sensing–based fast imaging method, SCOPE, was used to accelerate the MS‐T1ρ acquisition time to a total scan time of 3 minutes 31 seconds. A phantom experiment was conducted to assess the accuracy of MS‐T1ρ by comparing the T1ρ value obtained using MS‐T1ρ with the reference value obtained using the standard single‐slice T1ρ mapping method. In vivo scans of 13 volunteers were acquired prospectively to validate the robustness of MS‐T1ρ.ResultsIn the phantom study, the T1ρ values obtained with MS‐T1ρ were in good agreement with the reference T1ρ values (R2 = 0.9991) and showed high consistency throughout all slices (coefficient of variation = 2.2 ± 2.43%). In the in vivo experiments, T1ρ maps were successfully acquired for all volunteers with no visually noticeable artifacts. There was no significant difference in T1ρ values between MS‐T1ρ acquisitions and fully sampled acquisitions for all brain tissues (p‐value > .05). In the intraclass correlation coefficient and Bland‐Altman analyses, the accelerated T1ρ measurements show moderate to good agreement to the fully sampled reference values.ConclusionThe proposed MS‐T1ρ solution allows for high‐resolution whole‐brain T1ρ mapping within 4 minutes and may provide a potential tool for investigating neural diseases.

Chloroethylagelastatin A (CEAA) is an analogue of agelastatin A (AA), a natural alkaloid derived from a marine sponge. It is under development for therapeutic use against brain tumors as it has excellent central nervous system (CNS) penetration and pre-clinical therapeutic activity against brain tumors. Recently, AA was shown to inhibit protein synthesis by binding to the ribosomal A-site. In this study, we developed a novel virtual screening platform to perform a comprehensive screening of various AA analogues showing that AA analogues with proven therapeutic activity including CEAA have significant ribosomal binding capacity whereas therapeutically inactive analogues show poor ribosomal binding and revealing structural fingerprint features essential for drug-ribosome interactions. In particular, CEAA was found to have greater ribosomal binding capacity than AA. Biological tests showed that CEAA binds the ribosome and contributes to protein synthesis inhibition. Our findings suggest that CEAA may possess ribosomal inhibitor activity and that our virtual screening platform may be a useful tool in discovery and development of novel ribosomal inhibitors.

RXRA is an endothelial- and early stage–predominant therapeutic target for treating anthracycline-induced cardiotoxicity.

Abstract Although degenerative disc disease (DDD) and related low back pain (LBP) are growing public health problems, the underlying disease mechanisms remain unclear. An increase in the vascular endothelial growth factor (VEGF) levels in DDD has been reported. This study aimed to examine the role of VEGF receptors (VEGFRs) in DDD, using a mouse model of DDD. Progressive DDD was induced by anterior stabbing of lumbar intervertebral discs in wild type (WT) and VEGFR‐1 tyrosine‐kinase deficient mice ( vegfr‐1 TK−/ − ). Pain assessments were performed weekly for 12 weeks. Histological and immunohistochemical assessments were made for discs, dorsal root ganglions, and spinal cord. Both vegfr‐1 TK − / − and WT mice presented with similar pathological changes in discs with an increased expression of inflammatory cytokines and matrix‐degrading enzymes. Despite the similar pathological patterns, vegfr‐1 TK − / − mice showed insensitivity to pain compared with WT mice. This insensitivity to discogenic pain was related to lower levels of pain factors in the discs and peripheral sensory neurons and lower spinal glial activation in the vegfr‐1 TK − / − mice than in the WT mice. Exogenous stimulation of bovine disc cells with VEGF increased inflammatory and cartilage degrading enzyme. Silencing vegfr‐1 by small‐interfering‐RNA decreased VEGF‐induced expression of pain markers, while silencing vegfr‐2 decreased VEGF‐induced expression of inflammatory and metabolic markers without changing pain markers. This suggests the involvement of VEGFR‐1 signaling specifically in pain transmission. Collectively, our results indicate that the VEGF signaling is involved in DDD. Particularly, VEGFR‐1 is critical for discogenic LBP transmission independent of the degree of disc pathology.

Abstract Background Melphalan, prednisone and thalidomide (MPT) is a standard therapy for NDMM recognized worldwide based on a statistically significant advantage in overall survival (OS) and progression-free survival (PFS) vs. MP (Facon Lancet 2007; Fayers Blood 2011; NCCN 2013). The combination of lenalidomide and low-dose dexamethasone (Rd) increased OS with fewer adverse events (AEs) than treatment with lenalidomide and high-dose dexamethasone in NDMM pts (ECOG E4A03) (Rajkumar Lancet Oncol 2010). The FIRST trial is a multicenter, open-label, phase III trial comparing the efficacy and safety of Rd versus MPT in transplant-ineligible NDMM pts. Methods NDMM pts either ≥ 65 years of age, or not candidates for SCT were randomized to one of three arms: Rd in 28-day cycles until disease progression (Arm A), Rd in 28-day cycles for 72 weeks (18 cycles, Arm B), or MPT in 42-day cycles for 72 weeks (12 cycles, Arm C). Assessments by International Myeloma Working Group criteria were done after each cycle. Pts with renal impairment were enrolled; however, pts on dialysis were excluded. Starting doses of lenalidomide and dexamethasone were adjusted based on renal function and age, respectively. Melphalan starting dose was adjusted based on age, absolute neutrophil count, platelet count, and renal function; and thalidomide was adjusted for age. Dose adjustments were permitted for AEs. All pts were required to receive anti-thrombotic prophylaxis. Stratification factors included age, International Stage System, and country. The primary endpoint was a comparison of PFS in Arm A vs. Arm C. Secondary endpoints included OS, overall response rate (ORR), time to response, duration of response (DOR), safety, and quality of life (QOL). A preplanned additional analysis included time from randomization to second progression event or death (PFS2). The final preplanned analysis of independently adjudicated progressive disease (PD) events in Arm A vs. Arm C conducted after 960 events of death or PD, and an interim of OS in 64% of survival events (574/896 events) are presented in this abstract. Comparisons of PFS and all secondary endpoints, including OS for all three arms, will be presented at the meeting. Results A total of 1,623 pts were randomized 1:1:1 in three arms. As of today, 121 pts continue to receive lenalidomide on study (Arm A). The median age was 73 (40.0–92.0) years; 35% pts were aged ≥ 75 years; and 41% of pts had ISS stage 3 disease. After a median follow-up of 37 months, the trial met its primary endpoint (PFS), demonstrating a 28% reduction in risk of progression or death (HR=0.72; p= 0.00006). The preplanned interim analysis of OS demonstrated a 22% reduction in risk of death in favor of Arm A vs. Arm C (HR=0.78, p=0.01685); however, the pre-specified boundary (p<0.0096) was not crossed. All other secondary endpoints consistently showed improvement in favor of Arm A vs. Arm C; ORR (PR or better) 75% vs. 62% (p < 0.00001), DOR (HR=0.63; p<0.00001), and PFS2 (HR=0.78, p=0.0051). Relevant Grade 3/4 adverse events in Arm A vs. Arm C were neutropenia (28% vs. 45%), thrombocytopenia (8% vs. 11%), febrile neutropenia (1% vs. 3%), infection (29% vs. 17%), neuropathy (5% vs. 15%), and deep-vein thrombosis (5% vs. 3%). The incidence of secondary primary malignancies (SPM) was evaluated. Hematologic malignancies were 0.4% in Arm A vs. 2.2% in Arm C; the overall incidence of solid tumors was identical (2.8%). Conclusion Continuous treatment with the all oral doublet Rd significantly improved the primary endpoint of PFS compared with the standard triplet, MPT. All secondary endpoints support the clinical benefit of continuous Rd treatment. The safety profile of Rd was manageable, with reduced hematologic SPM compared to MPT. Disclosures: Facon: Celgene: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Off Label Use: Lenalidomide as treatment for NDMM. Dimopoulos:Celgene, Orthobiotech: Honoraria. Dispenzieri:Celgene, Millenium, Jansenn, Pfizer: Research Funding. Catalano:Celgene, Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Hulin:Janssen: Honoraria; Celgene: Honoraria. Cavo:Celgene, Janssen, Millennium, Onyx, Brystol Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees. Pinto:Mundipharma: Consultancy, Honoraria; Roche: Honoraria; Celgene: Consultancy, Honoraria. Weisel:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Ludwig:Celgene: Honoraria, Research Funding, Speakers Bureau. Bahlis:Celgene: Consultancy, Honoraria. Delforge:Celgene: Honoraria. Chen:Celgene: Consultancy, Research Funding; Lundbeck: Consultancy; Johnson & Johnson: Consultancy, Research Funding; Roche: Honoraria; GlaxoSmithKline: Research Funding. Oriol:Celgen: Consultancy. White:Celgene: Honoraria, Research Funding. Binder:Celgene: Research Funding. Anderson:Acetylon, OncoPep: Equity Ownership; Celgene, Onyx, Sanofi Aventis, Gileod: Consultancy. Moreau:Celgene Corporation: Honoraria, Speakers Bureau. Attal:Janssen: Lectures, Lectures Other, Membership on an entity’s Board of Directors or advisory committees; Celgene Corporation: Lectures Other, Membership on an entity’s Board of Directors or advisory committees. Knight:Celgene: Employment, Equity Ownership. Chen:Celgene: Employment. Van Oostendorp:Celgene: Employment. Jacques:Celgene: Employment. Ervin-Haynes:Celgene: Employment, Patents & Royalties. Benboubker:Celgene: Consultancy.

Abstract Abstract 3513 To identify targets for rational combination therapies with 5-Azacytidine (5-Aza) in myeloid malignancies, we utilized high-throughput RNA-interference (RNAi) viability screening. A siRNA library targeting 572 kinases and a custom collection of 289 putative cancer targets, including cell cycle and apoptosis regulatory genes, were screened alone and in combination with 5-Aza in TF-1 and ML-2 myeloid leukemia cell lines to identify synergistic interactions in reducing cell viability. Of the 572 kinases that were individually silenced, less than 1% significantly increased sensitivity to 5-Aza. The kinase library was also screened in combination with 5-Aza in a third myeloid cell line, THP-1, confirming that few kinases sensitize to 5-Aza when inhibited. While few kinases sensitized to 5-Aza, the anti-apoptotic Bcl-2 family of genes emerged as potent sensitizers to 5-Aza from RNAi screens. Therefore, silencing by siRNA of BCL-XL, BCL-2, BCL-W, MCL-1 and BFL-1 was evaluated in combination with 5-Aza treatment in an expanded panel of myeloid cell lines including TF-1, HEL, THP-1, ML-2 and MDS-L. BCL-XL validated as a vulnerability and potent sensitizer to 5-Aza in erythroid leukemia cell lines TF-1 and HEL, whereas MCL-1 was a strong vulnerability in the monocytic leukemia cell line THP-1 and also a moderate sensitizer to 5-Aza in ML-2, THP-1, TF-1 and HEL. Published proteomics data from our group indicate that M6 and M7 leukemias exhibit higher levels of BCL-XL, while additional unpublished data suggest elevated levels of MCL-1 in M4 and M5 leukemias, supporting our functional observations. Additionally, data from the public database Oncomine suggest that BCL-XL expression is elevated in M6 and M7 leukemias while MCL-1 shows a trend towards elevation in M4 and M5 leukemias. Based on RNAi screening results, siRNA validation experiments and proteomic/mRNA expression data, we evaluated the BCL-2/BCL-XL/BCL-W inhibitor ABT-737 in combination with 5-Aza. ABT-737 resulted in dose-dependent sensitization to 5-Aza in all AML-derived cell lines examined (including M7, M6, M5, M4 and M2 FAB subtypes) and in the MDS cell line MDS-L; however, no sensitization was observed in the CML cell line K562. In extensive ex vivo experiments with 17 primary specimens, potent synergy between 5-Aza and ABT-737 was observed in AML, MDS and MPN samples, but not in most CML samples examined. Calculations with CalcuSyn software demonstrate synergy, with combination index values as low as 0.2, between 5-Aza and ABT-737 both ex vivo and in vitro. The combination of 5-Aza with ABT-737 resulted in substantial induction of apoptosis, measured by the induction of cleaved caspase 3 in TF-1 and HL-60 cells, as compared to either compound alone. Interestingly, although siRNA silencing of MCL-1 in combination with 5-Aza was potent across several cell lines, and silencing of BCL-XL preferentially in an erythroid differentiation background, ABT-737 with 5-Aza sensitized across a variety of cell lines and all myeloid primary specimens ex vivo. We suggest that inhibition of anti-apoptotic Bcl-2 family members is a most promising rational combination strategy with 5-Aza for the treatment of leukemias. Our results also highlight the potential utility of more specific anti-apoptotic Bcl-2 family inhibitors in the lineage-specific treatment of myeloid malignancies. Disclosures: Off Label Use: AraC in AML. Experimental Agent MK1775. Mesa:Incyte: Research Funding; Lilly: Research Funding; SBio: Research Funding; Astra Zeneca: Research Funding; NS Pharma: Research Funding; Celgene: Research Funding.

Abstract Introduction: Cytogenetic abnormalities in patients (pts) with multiple myeloma (MM) are of prognostic importance and can be associated with poor outcomes (Bergsagel, Blood, 2011). The FIRST trial is a pivotal phase 3 study with the largest data set in transplant-ineligible pts with newly diagnosed MM (NDMM). This subanalysis evaluates the impact of cytogenetics on outcomes in transplant-ineligible pts with NDMM continuously treated with lenalidomide plus low-dose dexamethasone until disease progression (Rd continuous). Methods: Transplant-ineligible pts with NDMM were randomized to 1 of 3 treatment arms: Rd continuous, Rd18 (Rd for 18 cycles [72 weeks]), or melphalan-prednisone-thalidomide (MPT; for 12 cycles [72 weeks]). Cytogenetics were assessed using fluorescence in situ hybridization. Pts were categorized into cytogenetic risk groups according to International Myeloma Working Group criteria. High-risk cytogenetics included del(17p), t(4;14), and t(14;16); all other pts were categorized as non-high risk. The primary endpoint was progression-free survival (PFS; primary comparators were Rd continuous vs MPT), and key secondary endpoints were overall survival (OS), overall response rate (ORR), and safety. Results: A total of 762 of 1623 pts from the intent-to-treat population had validated cytogenetic profiles, with 142 pts in the high-risk group and 620 pts in the non-high-risk group. Baseline characteristics were well balanced across cytogenetic risk groups (Table 1). The median follow-up for OS was 40.2 months for the 762 pts in this analysis (data cutoff, March 03, 2014). In the non-high-risk group, median duration of treatment was 19.4 months with Rd continuous and 16.6 months with both Rd18 and MPT. In the high-risk group, median duration of treatment was 10.0 months with Rd continuous, 8.2 months with Rd18, and 12.0 months with MPT. Rd continuous treatment resulted in a 24% reduced risk of death or progression compared with MPT and an even greater 32% reduced risk in pts without high-risk cytogenetics (Table 2). In non-high-risk pts, median PFS was 31.1 months with Rd continuous compared with 21.2 and 24.9 months with Rd18 and MPT, respectively (Figure). However, in high-risk pts, the observed numerical median PFS favoring Rd18 is mainly due to small pt numbers influenced by long runners (n = 5), and the greatly overlapping 95% CIs from all 3 arms show the difference is likely to be minimal. Rd continuous treatment resulted in a 28% reduced risk of death vs MPT overall and a 34% reduced risk in pts without high-risk cytogenetics. OS was similar across treatment arms for high-risk pts. ORRs in all cytogenetic risk groups favored Rd continuous vs MPT. In pts with high-risk cytogenetics, higher-quality responses were also observed with Rd continuous vs MPT treatment. Similar results were seen with Rd continuous compared with Rd18, although OS and ORR benefits overall and in pts without high-risk cytogenetics were not as pronounced. In all 3 treatment arms, adverse events were consistent across cytogenetic risk groups. Conclusions: Rd continuous treatment resulted in PFS and OS benefits vs MPT in pts with validated cytogenetic profiles. This was largely due to PFS and OS improvements in pts without high-risk cytogenetics. In the high-risk group, the longest PFS was observed with Rd18 treatment and OS was similar across treatment arms. Despite being on the continuous vs fixed duration treatment arm, high-risk pts on Rd continuous received a shorter duration of treatment than those on MPT, which may explain why PFS favored MPT vs Rd continuous. Higher response rates were observed with Rd continuous vs MPT, regardless of cytogenetic risk, and greater quality responses were observed in pts with high-risk cytogenetics. The safety profile of Rd continuous was manageable and consistent between cytogenetic risk groups. Results support Rd continuous as a standard treatment option for pts with NDMM who are ineligible for transplant, especially those without high-risk cytogenetics. Additional PFS and OS benefits may be achieved in pts with high-risk cytogenetics when Rd continuous is used as a backbone for combination therapy with a novel agent. Promising activity in pts with high-risk cytogenetic abnormalities has been demonstrated using this approach (Lonial et al, N Engl J Med, 2015; Stewart et al, N Engl J Med, 2015). Disclosures Hulin: Celgene Corporation: Honoraria; Janssen: Honoraria; Amgen: Honoraria; Bristol Myers Squibb: Honoraria. Dimopoulos:Celgene: Honoraria; Onyx: Honoraria; Novartis: Honoraria; Janssen-Cilag: Honoraria; Amgen: Honoraria; Janssen: Honoraria; Genesis: Honoraria. Reece:Lundbeck: Honoraria; Amgen: Honoraria; Merck: Research Funding; Bristol-Myers Squibb: Research Funding; Janssen-Cilag: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Millennium Takeda: Research Funding; Otsuka: Research Funding. Catalano:Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Celgene Corporation: Consultancy, Honoraria. Pinto:Takeda: Honoraria, Research Funding; Celgene Corporation: Honoraria; Spectrum: Honoraria. Ludwig:Takeda: Research Funding; Celgene Corporation: Honoraria, Speakers Bureau; Onyx: Honoraria, Speakers Bureau; Bristol Myers Squibb: Honoraria, Speakers Bureau; Janssen Cilag: Honoraria, Speakers Bureau. Bahlis:Celgene Corporation: Honoraria, Research Funding. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria. Moreau:Takeda: Other: Adboard; Janssen: Other: Adboard; Novartis: Other: Adboard; Amgen: Other: Adboard; Celgene: Honoraria, Other: Adboard. Qiu:Johnson & Johnson: Speakers Bureau; Celgene Corporation: Speakers Bureau; Roche: Speakers Bureau. Schots:Celgene Corporation: Research Funding. Marek:Celgene Corporation: Employment, Equity Ownership. Chen:Celgene Corporation: Employment, Equity Ownership. Yiu:Celgene Corporation: Employment, Equity Ownership. Ervin-Haynes:Celgene Corporation: Employment. Facon:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millenium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pierre Fabre: Membership on an entity's Board of Directors or advisory committees.

Abstract Introduction Primary CNS lymphoma (PCNSL) is a diffuse large B-cell lymphoma (DLBCL), predominantly of non-germinal center (non-GC) subtype. Currently available treatments remain disappointed with a non-optimal complete remission rate and a high relapse rate. As such, novel therapeutic agents are urgently needed. Constitutive activation of the NF-kB pathway via mutations in B cell receptor (BCR) (CD79B) and mutation of MYD 88 and TBL1XR1 pathways plays an important role in PCNSL. Ibrutinib, an inhibitor of BCR signaling, has been found to have significant therapeutic activity in relapsed or refractory non-CNS non-GC DLBCL that display similar NFKB mutational patterns. We have performed a CNS pharmacokinetic (PK) study of Ibrutinib and evaluated its therapeutic activity against CNS lymphoma (CNSL) in murine models. Methods CNS PK study in mice - The first study was done in a CNSL model created by intracerebral implantation of luciferase-transfected lymphoma cells (MC116) in nude mice in comparison to healthy nude mice without lymphoma cell implantation (N=5 per group). Once the tumor activity was confirmed by bioluminescence imaging in CNSL group, all the mice were treated with ibrutinib 12 or 30 mg/kg/day by oral gavage (OG) for 5 days. Blood and brain were collected 2 hours after the last dose. Ibrutinib was measured by ultra performance liquid chromatography with tandem mass spectrometry. Cerebro-spinal fluid (CSF) PK study in Sprague-Dawley (SD) rats - Ibrutinib 50 mg/kg single dose was given by OG (N=6). Microdialysates of CSF were collected from a ventricle of the brain and a carotid artery via microdialysis catheters every hour for 13 hours post-treatment. Drug concentrations were determined by capillary electrophoresis with laser detection. Preclinical therapeutic evaluation - A murine CNS lymphoma model was created by intracerebral injection of the OCI-Ly10 non-GC DLBCL cells, which were confirmed to have CD79 A and MYD 88 mutations and show high sensitivity to Ibrutinib in vitro. Animals were sacrificed if they showed severe clinical signs or symptoms or >20% weight loss. Ibrutinib 30 mg/kg was administered by OG daily starting at day 7 post-implantation and until the last sacrifice in the control group treated with vehicle. Therapeutic efficacy was determined by Kaplan-Meier survival analysis using date of sacrifice as endpoint. Results CNS PK study in mice - 1) Ibrutinib levels in plasma, normal brain tissue and brain-tumor were analyzed. Ibrutinib was not detected in healthy control mice treated with vehicle. In healthy mice treated with ibrutinib (12 mg/kg and 30 mg/kg), Ibrutinib mean plasmatic levels were 9.31 (±3.56) ng/mL and 42.2 (±20.9) ng/mL respectively. At 12 mg/kg dose level, ibrutinib was detectable but not quantifiable in brain tissue. After treatment with 30 mg/kg,iIbrutinib mean levels were 0.0077 (±0.0053) and 0.0071 (±0.00483) ng/g tissue in right and left cerebral hemispheres respectively. In CNSL mice treated with ibrutinib (12 mg/kg and 30 mg/kg), Ibrutinib mean plasmatic levels were 12.03 (±9.04) ng/mL and 42.64 (±42.04) ng/mL respectively, brain ibrutinib levels were 0.008 (±0.005) and 0.007 (±0.003) ng/g tissue for tumor-bearing right hemisphere and non-tumor bearing left hemisphere respectively for 30 mg/kg, while brain ibrutinib level was detectable but not quantifiable at 12mg/kg. CSF PK study in SD rats - CSF penetration of ibrutinib was determined to be ~5.2 % as calculated by area under the curve (AUC) ratio of CSF and blood samples. Cmax in CSF is ~0.35 uM, which is significantly higher than published EC50 concentrations for DLBCL cell lines. Preclinical therapeutic evaluation -Ibrutinib showed an excellent therapeutic activity with significant prolongation of survival (p=0.0137) (fig 1). At a median follow-up of 112 days, the median survival in treated CNSL group is 56 days compared to 33 days in the control group. At the end of the follow-up 33% of animals in treated CNSL group are still alive without treatment. Conclusion Ibrutinib achieves high concentrations in brain and CSF in murine CNS PK models with promising single-agent preclinical therapeutic activity against CNS lymphoma in a murine model. Further studies to develop combination regimen are ongoing. Figure Figure. Disclosures Soussain: Roche: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding.

Abstract Hypomethylating agents have changed treatment and outcomes in MDS and are active in AML patients. However a significant number of patients do not respond to 5-Azacytidine or Decitabine and there is no clinically validated assay that predicts response to 5-Azacytidine (5-Aza) or Decitabine (DAC). Clinical parameters and recently TET2 mutations have been identified as genetic predictors of response to 5-Aza. Several recent papers report on the possible predictive value of anti-apoptotic proteins, for example BCL2L10. In previous genome-scale RNA-interference (RNAi) screens anti-apoptotic BCL-2 family members, most potently BCL-XL, were identified as top targets whose down-regulation sensitize to 5-Aza. Results were confirmed with the BCL-XL and BCL-2 inhibitor ABT-737, as well as the BCL-2-specific inhibitor ABT-199, both of which sensitized myeloid cells to 5-Aza. Although it is expected that interfering with anti-apoptotic genes would sensitize to hypomethylating similar to cytotoxics, few of the other >900 genes from our RNAi sensitizer screens, including few of the 571 kinases, sensitized to 5-Aza as potently as inhibition of BCL-XL. Thus BCL-2 family members constitute an especially potent context to modulate the activity of 5-Aza. To assess if protein expression of the top targets BCL-XL, BCL-2 or MCL-1 correlate with and may explain a preferential sensitization, 577 primary AML samples were examined by Reverse Phase Protein Array (RPPA). There was substantial overlap of expression across and within FAB subgroups and cytogenetics, and expression of neither of these genes/proteins could explain the observed effects. This may be expected due to functional redundancies amongst the different anti-apoptotic BCL-2 family members, as well as distinct, yet overlapping binding affinities between anti- and pro-apoptotic BCL-2 proteins and BH3 peptides. Thus, we next aimed to functionally interrogate the overall balance of pro- and anti-apoptotic BCL-2 family members, also described as “primedness” by BH3 profiling. This assay uses peptides derived from pro-apoptotic BH3-only proteins to determine the capacity of cells to initiate apoptosis in response to pro-apoptotic BH3 molecules [anti-apoptotic BCL-2 molecules like BCL-XL and BCL-2 bind and inhibit BH3-only molecules]. This assay is a broad functional readout that incorporates many parameters including the consequences of varying BCL2L10 expression. To establish a proof-of-concept that the functional interrogation of specific apoptotic thresholds may be an optimal readout for 5-Aza response, we first assessed a broad panel of 13 AML-derived cell lines by BH3 profiling and in parallel 5-Aza drug dose response experiments. Several BH3 profiling metrics, including NOXA plus BIM, significantly correlated with 5-Aza sensitivity in vitro (Figure 1). Next, to correlate actual clinical responses to 5-Aza with BH3 profiling, specimens from 28 AML, MDS and MDS/MPN overlap patients treated with 5-Aza or DAC-based regimens and for whom clinical outcome was available, were assayed in BH3 assays. In the clinic, the best BH3 metrics were combined values from NOXA and BIM peptides similar to cell lines. NOXA plus BIM discriminated clinical responses defined as achieving any response (sensitive) versus a patient being resistance/refractory to 5-Aza/DAC based regimens with statistical significance (Mann-Whitney two-tailed p = 0.001). This was true for the entire group of patients (5-Aza- and DAC- regimens) with a Receiver Operating Characteristic (ROC) of an AUC=0.875, with a further increase for patients treated only with 5-Aza based regimens with an AUC=0.95.Figure 1BH3 metrics distinguish cell lines with higher EC 50 (> 2uM) from those with lower EC50 (generally < 1uM).Figure 1. BH3 metrics distinguish cell lines with higher EC 50 (> 2uM) from those with lower EC50 (generally < 1uM). In conclusion, due to significant expression overlap and functional redundancies of the BCL-2 family proteins, expression does not correlate with 5-Aza clinical or in vitro responses. However, BH3 profiling as a general functional readout of apoptotic primedness, significantly discriminated responses in patients as well as in vitro. Thus, BH3 profiling incorporates the entirety of pro- and anti-apoptotic molecules (including BCL2L10) and is promising to predict responses to hypomethylating agents. We propose to prospectively validate BH3 profiling as a predictive biomarker assay for 5-Aza or DAC based regimens. Disclosures: Pierceall: Eutropics: Employment, Equity Ownership. Lena:Eutropics: Employment, Equity Ownership. Cardone:Eutropics: Employment, Equity Ownership. Elashoff:Eutropics: Employment. Blake:Noel Blake: Employment, Equity Ownership.

Abstract Cold agglutinin disease (CAD) is an autoimmune hemolytic anemia characterized by the presence of autoantibodies (cold agglutinins) that bind to red blood cells (RBC) at low temperatures. Cold agglutinin binding to RBCs results in antibody-mediated classical complement activation with deposition of complement C4 and C3 fragments onto the RBC surface. Complement-opsonized RBCs are removed from the circulation by macrophage-driven phagocytosis in the liver or spleen and via intravascular hemolysis following assembly of the terminal lytic complex (C5b - C9), together resulting in clinical anemia. C1q is the initiating molecule for cold agglutinin-mediated complement activation on the surface of human RBCs. We hypothesized that directly blocking C1q recruitment onto cold agglutinin-sensitized RBCs will prevent complement activation and opsonization and reduce hemolysis. To this end, we have developed a humanized monoclonal antibody (ANX005) that binds with high-affinity (~10 pM) to C1q and blocks classical complement activation & hemolysis in an in vitro sheep RBC assay. We evaluated the impact of ANX005 on hemolysis and complement deposition on human RBCs that were pre-sensitized with sera from CAD subjects. ANX005 showed a dose-dependent reduction in hemolysis using both individual and pooled CAD sera as the source of cold agglutinin. We further demonstrated that C1q blockade led to a robust reduction in C4 and C3 fragment deposition onto human RBCs. These results demonstrate that C1q inhibition is an effective way to impede C4 and C3 activation and downstream assembly of the lytic complex in sera from CAD patients, and support the clinical development of ANX005 in CAD and other antibody-mediated diseases. Disclosures Gertz: Prothena Therapeutics: Research Funding; Sandoz Inc: Honoraria; NCI Frederick: Honoraria; Celgene: Honoraria; Med Learning Group: Honoraria, Speakers Bureau; Research to Practice: Honoraria, Speakers Bureau; Alnylam Pharmaceuticals: Research Funding; Novartis: Research Funding; Ionis: Research Funding; Annexon Biosciences: Research Funding; GSK: Honoraria. Qiu:Annexon Biosciences: Employment, Equity Ownership. Kendall:Annexon Biosciences: Employment, Equity Ownership. Saltarelli:Mallinckrodt: Equity Ownership; Abbvie: Equity Ownership; Annexon Biosciences: Employment, Equity Ownership, Patents & Royalties. Yednock:Annexon, Inc: Employment, Equity Ownership. Sankaranarayanan:Annexon Biosciences: Employment, Equity Ownership.