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The benefits of caplacizumab in acute immune mediated TTP (iTTP) are well established. We identified a delay in normalisation of ADAMTS13 activity (>30%) in a subgroup of caplacizumab treated patients which was not evident in the pre-caplacizumab era. Caplacizumab treated patients (n=64) achieved ADAMTS13 activity >30% at median 31 days post PEX, compared to 11.5 days in the non-caplacizumab group (n=50, p=0.0004). 18/64 (28%) caplacizumab treated patients had an ADAMTS13 activity 30% (median 139 days after completing PEX). 18/64 (28%) of patients receiving extended caplacizumab (31-58 days) failed to achieve ADAMTS13 activity >30% at time of stopping caplacizumab compared to 4/47 (8.5%) of historical controls at similar timepoint (30+28 days, p30% within 30+28 days was 6 times more likely in caplacizumab treated patients (OR 6.3, p=0.0006). ADAMTS13 antigen level

Refractory acute myeloid leukemia (AML), defined as failure of 2 cycles of induction therapy at diagnosis or of 1 cycle at relapse, represents a subgroup with poor clinical outcomes. In our transplant cohort, the 5-year overall survival in this subgroup was 16% (Ganapule at al. JGO 2017). Haploidentical natural killer cell (NK) therapy is a strategy that is being explored in refractory malignancies. Our in-vitro and animal model data suggest that exposure to arsenic trioxide (ATO) results in enhanced NK cytotoxicity (Alex AA et al. Front. Immunol 2018). Historically, at our center, patients with refractory AML have been treated with cytoreductive therapy (FLAG ± idarubicin or mitoxantrone + etoposide for 3 to 5 days) followed by 1-week rest and then a reduced-intensity transplant with fludarabine + melphalan conditioning while in peak cytopenia. From February 2019, we initiated a phase II single arm clinical trial (CTRI/2019/02/017505) enrolling patients with refractory AML planned for a stem cell transplant in peak cytopenia. Patients received CD56-positive cells from an HLA haploidentical related family donor (other than the stem cell donor, wherever feasible) following cytoreductive chemotherapy. The NK cell donor preference strategy included presence of KIR ligand mismatch, greater number of KIR B motifs (or the B score), lower donor age, and negative donor specific antibodies tested using flowcytometry crossmatch (Figure 1a). CD56-positive selection was done using CliniMACS prodigy system. This was followed by overnight incubation of the CD56 positive cells in autologous plasma with 2 micromolar ATO and 500 U/mL of interleukin-2. The CD56 positive cells were then infused to the patient 1-day after the completion of cytoreductive chemotherapy. This was followed by a reduced intensity stem cell transplant (Figure 1b). The primary outcome variable was 1-year relapse free survival. From February 2019, 14 patients with median age 28 years (IQR: 15.75-31.5) were enrolled in this trial. Six were females. Six had primary-refractory AML while 8 had relapsed-refractory AML. The cytoreductive chemotherapy was FLAG ± idarubicin (n=7), Mitoxantrone + Etoposide (n=6) and GCLAC (n=1). The median blast percentage on flowcytometry MRD testing prior to NK infusion was 15.9% (IQR: 9.1%-54.5%) (n=11). The median B score for the NK cell donors was 2 (IQR: 1-3). The median age of the NK cell donor was 43 years (IQR: 36-49.5). KIR ligand mismatch with the patient was noted in 2 donors. The median CD56-cell dose infused was 46.16 x 10 6/kg (IQR: 25.06-70.36) (Figure 1c). Pre-defined release criteria, including sterile cultures, and endotoxin negativity were met in all cases. There was no infusion related toxicity. The median blast percentage on flowcytometry MRD testing done following NK cell infusion was 11.9% (IQR: 4.9%-47.6%) (n=8). One patient withdrew consent after NK cell infusion and did not undergo transplant. For the remaining 13 patients, the stem cell donor was HLA matched (n=4), HLA 9/10 matched (n=1) or HLA haplomatched (n=8). The median CD34 cell dose infused was 10 x 10 6/kg (IQR:7.51-11.6). Five (38.5%) patients died of immediate post-transplant complications (sepsis (n=3) on days 1, 2 and 28, cerebral venous sinus thrombosis (n=1) on day 1 in a patient treated with hormonal contraceptives for menorrhagia, and veno-occlusive disease (n=1) on day 15 in a patient undergoing a second transplant) while 2 (15.4%) did not engraft (both subsequently died of infective complications following engraftment post-second transplant). Of the remaining 6 (46.2%) patients who engrafted and survived beyond 1 month of the transplant, the day 28 post-transplant MRD was negative for 5 patients while it was positive in 1 patient (0.13%). On follow up, 2 (15.4%) patients developed disease relapse (on days 54 and 218 respectively) and died. The remaining 4 (30.8%) patients are alive and relapse free at last follow up (mean follow up of surviving patients is 16 months). One patient received a CD34 cell boost on day 96 (cell dose - 8.55 x 10 6/kg) for poor graft function. For the entire cohort, the estimated 1-year event free survival is 28.8% ± 13.1%(Figure 1d). Whereas, acute GVHD was noted in 3 patients (50%; out of 6 evaluable patients) and chronic GVHD was noted in 3 patients (50%; out of the 6 evaluable patients). Thus, haploidentical NK cell therapy as an adjunct to transplant is safe and merits further evaluation in patients with AML. Figure 1 Figure 1. Disclosures Mathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.

Background: Use of peripherally inserted central catheters (PICCs) has grown rapidly in patients with hematologic malignancies. Studies demonstrating the safety of PICCs in hematologic malignancies, however, are largely single-center, retrospective designs focused on the outpatient setting. Little is known about inpatient PICC use in patients with hematologic malignancies and how such use varies across hospitals. Methods: Data was prospectively collected between November 2013 and December 2019 from a cohort of patients admitted at one of 42 Michigan hospitals participating in the Michigan Hospital Medicine Safety Consortium (HMS). Patients had a diagnosis of a hematologic malignancy and had a PICC placed during their hospital stay. Adult medical patients admitted to a general ward or intensive care unit were eligible for data collection and data were collected from the medical record using a standardized template. The indications for PICC placement, catheter characteristics, and associated complications from 17 hospitals with more than 25 discrete patients with hematologic malignancy were included in a comparative analysis. Major complications were defined as central line associated bloodstream infection (CLABSI), catheter occlusion, and venous thromboembolism (VTE). Differences across hospitals were tested using the Kruskal-Wallis test for continuous variables and Pearson chi-square test for categorical variables. Results: A total of 2092 PICCs placed in 1798 patients were included in the analysis with a median (IQR) dwell time of 15 (6-30) days. Most patients were male (n=1242, 59.4%), white (n=1720, 82.2%), and 50 to 69 years old (n=950, 45.4%). Patients were primarily admitted to large hospitals (≥375 beds: n=1429, 68.8%) that were teaching centers (n=1600, 76.5%) in metropolitan locations (n=2000, 95.6%). Leukemia (n=752, 36.0%) and non-Hodgkin's lymphoma (n=409, 19.6%) were the most represented malignancies. The most common primary indication for PICC placement was administration of chemotherapy (n=1180, 56.4%). The majority of PICCs were double lumen (n=1457, 69.6%), most often placed by vascular access nurses (n=1520, 72.7%). A concurrent central venous catheter was present at the time of PICC placement in 12.2% (n=264) of patients. A major complication event occurred in over 1 in 4 PICCs placed (n=562, 26.9%). Catheter occlusion occurred in 17.8% (n=372), CLABSI in 8.2% (n=171), and VTE in 3.8% (n=80) of PICCs respectively. There was wide variation in PICC indications, characteristics, and outcomes across hospitals. Placement of PICCs for chemotherapy varied from 33.3% to 85.3% (p Conclusion: Appropriate venous access is critical to the care of patients with hematologic malignancy. This study demonstrates wide variation in the practice patterns and outcomes for PICCs in patients with hematologic malignancies across hospitals in Michigan. Further work is necessary to further understand and improve decision making around choosing vascular access in this vulnerable population. Figure 1 Figure 1. Disclosures Zhang: SIMR, Inc: Ended employment in the past 24 months, Research Funding; AmerisourceBergen: Current Employment. Sood: Bayer: Consultancy.

Background: DNA methyltransferase inhibition (DNMTi) with hypomethylating agents (HMA), azacitidine (AZA) or decitabine (DAC), remains the mainstay of therapy for most high-risk Myelodysplastic syndrome (MDS) patients. However, only 40-50% of MDS patients achieve clinical improvement with DNMTi. Previously, combinations of HMA and histone deacetylase (HDAC) inhibitors have been explored in MDS with varying clinical outcomes. However, the heterogeneity of genomic aberrations in MDS portend widely divergent responses from HDAC inhibition, implying that a predictive clinical decision support tool could select patients most likely to benefit from this combination. We explored the molecular basis of observed clinical response in a group of patients treated with DAC and Valproic-Acid (VPA). Method: 16 MDS patients with known clinical responses to DAC + VPA were selected for study from the Cellworks patient repository. The aberration and copy number variations from individual cases served as input into the Computational Omics Biology Model, a computational multi-omic biology software model largely created using literature sourced from PubMed, to generate a patient-specific protein network map. Disease biomarkers unique to each patient were identified within these maps. The Cellworks Biosimulation Platform has the capacity to biosimulate disease phenotypic behavior and was used to create a patient-specific disease model. Biosimulations were then conducted on each patient-specific disease model to measure the effect of DAC + VPA according to a cell growth score. This score was comprised of a composite of cell proliferation, viability, apoptosis, metastasis, and other cancer hallmarks. Biosimulation of drug response was conducted to identify and predict therapeutic efficacy. Results: In the biosimulation, VPA is a relatively weak HDAC inhibitor, but it also inhibits GSK3B and in turn increases beta-catenin (CTNNB1) levels. Additionally, monosomy 7 associated with loss of CAV1, HIPK2, and TRRAP also causes high CTNNB1, thereby further contributing to drug resistance. Biosimulation correctly identified that 7 of 8 patients with these genomic findings were clinical non-responders (NR) to VPA, indicating that CTNNB1 status is likely to predict treatment failure from the VPA + HMA combination in this disease. Notably, CTNNB1 levels have been reported to foster an immune-evasive tumor microenvironment resistant to CTL activation. By contrast, high levels of c-MYC predict response to VPA + HMA combination. VPA inhibits MYC transcription and thereby reduces MYC-induced downregulation of p21 through CKS1B. Additionally MYC is a transcriptional regulator of DNMT1 which is degraded after hyperacetylation induced by HDAC3 inhibition suggesting that VPA also enhances DNMT1 turnover. One patient analyzed had trisomy 8 resulting in c-MYC over-expression and responded to HMA + VPA. Additionally, other aberrations enhancing c-MYC transcription such as copy number variant (CNV) loss of MXI1, HHEX, FBXW7, SMAD7 or CNV gain of BRD4, BCL7B led to high clinical response to the combination (Table 1). By comparison to the CTNNB1-driven subset, the impact of VPA on CTNNB1 in the MYC-dominant disease network did not negate the benefit of VPA for these patients. Additionally, the inhibition of GSK3B by VPA leading to diminished FBXW7 and less ubiquitin-mediated turnover of c-MYC was not sufficient to overcome the inhibition of MYC transcription and HDAC3i-mediated turnover. Immune activation has become a recognized mechanism of responsiveness to HMA. However, among patients with upregulated CTNNB1, VPA is likely to further decrease response to treatment. By contrast, among MYC-driven cancers that are typically immune-evasive, VPA appears to be a vital mechanism of overcoming MYC-driven immune evasion. Conclusion: Signaling pathway consequences related to CTNNB1 and c-MYC upregulation predict response to DAC + VPA. Although HMA plus HDAC inhibition can be generally beneficial for MDS, variable mechanisms of action among various HDAC inhibitors and unique patient disease characteristics should be considered for optimal treatment selection. Finally, CTNNB1 emerged from the Cellworks biosimulations as a therapeutically relevant target in MDS that determines whether VPA synergizes or antagonizes the effect of other agents in this challenging subtype of MDS. Figure 1 Figure 1. Disclosures Castro: Caris Life Sciences Inc.: Consultancy; Omicure Inc: Consultancy; Cellworks Group Inc.: Current Employment; Exact sciences Inc.: Consultancy; Guardant Health Inc.: Speakers Bureau; Bugworks: Consultancy. Kumar: Cellworks Group Inc.: Current Employment. Grover: Cellworks Group Inc.: Current Employment. Patil: Cellworks Group Inc.: Current Employment. Kapoor: Cellworks Group Inc.: Current Employment. Agrawal: Cellworks Group Inc.: Current Employment. Sauban: Cellworks Group Inc.: Current Employment. Prasad: Cellworks Group Inc.: Current Employment. Basu: Cellworks Group Inc.: Current Employment. Suseela: Cellworks Group Inc.: Current Employment. Kumar: Cellworks Group Inc.: Current Employment. Nair: Cellworks Group Inc.: Current Employment. Kumari: Cellworks Group Inc.: Current Employment. Pampana: Cellworks Group Inc.: Current Employment. Ullal: Cellworks Group Inc.: Current Employment. Azam: Cellworks Group Inc.: Current Employment. Prasad: Cellworks Group Inc.: Current Employment. Amara: Cellworks Group Inc.: Current Employment. Sahu: Cellworks Group Inc.: Current Employment. Raveendaran: Cellworks Group Inc.: Current Employment. Veedu: Cellworks Group Inc.: Current Employment. Mundkur: Cellworks Group Inc: Current Employment. Patel: Cellworks Group Inc.: Current Employment. Christie: Cellworks Group Inc.: Current Employment. Macpherson: Cellworks Group Inc.: Current Employment. Howard: Servier: Consultancy; Cellworks Group Inc.: Consultancy; Sanofi: Consultancy, Other: Speaker fees.

In the setting of clinical trials, high cure rates have been reported for acute promyelocytic leukemia (APL) with the chemotherapy-free combination of arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) for low-intermediate risk disease, and with the addition of gemtuzumab ozogamicin or minimal anthracycline for high-risk disease. Despite clear in-vitro synergy between ATO and ATRA, most clinical trial protocols in APL have not used these drugs concurrently beyond induction. There is limited real world data on the use of chemotherapy-free combination of ATO and ATRA with minimal anthracycline use in the treatment of APL, especially in the high risk group. We did a retrospective analysis of the clinical outcomes of patients with newly diagnosed APL treated at our center from January 2015 to May 2020 using concurrent ATO, ATRA and minimal anthracycline in a risk stratified manner. The data was frozen and analyzed as on 1st June 2021. During the study period, 167 patients were diagnosed to have APL at our hospital. Of these, we excluded 5 patients who had presented with relapsed disease and 28 patients who were treated with single agent ATO. The remaining 134 patients were treated with a uniform protocol as summarized in Figure 1a. Figure 1b shows the KM plot for EFS (2 year: 90.8±2.5%) for these 134 patients. For analysis of regimen safety and efficacy, we excluded patients who wished to pursue treatment elsewhere within the first 1 week of treatment (n=7), and patients who had severe infections or life-threatening bleeding at presentation or before therapy initiation and subsequently died (n=4). Thus, a total of 123 patients with newly diagnosed APL were included for further analysis. The median age was 35 years (IQR: 24 to 45 years). Forty-six (37.4%) were females. The median duration of symptoms prior to admission was 14 days (IQR: 7 to 28 days). Sixty-one (49.6%) had high risk APL while the remaining 62 (50.4%) had low-intermediate risk APL. During induction, 28 (22.7%) patients had major bleeding while 5 (4%) patients developed major thrombosis. Sixty-seven (55%) patients had at least one documented infection during induction therapy. The median number of packed red cell concentrates, fresh frozen plasma, platelet rich concentrates and cryoprecipitates transfused during induction therapy was 5 (IQR: 3 to 6.5), 10 (IQR: 3 to 30), 40 (IQR: 23 to 55) and 6 (IQR: 0 to 18) respectively. Nineteen (15.4%) patients developed differentiation syndrome; all were treated with steroids, 8 required transient cessation of treatment and 6 required intensive care. Eight (6.5%) patients died during induction. During induction, grade 3 hepatotoxicity was noted in 8 (6.5%) patients, 10 (8.1%) patients had ATRA related headache or benign intracranial hypertension of which 5 required transient cessation of ATRA, while 11 (8.9%) patients had transient QTc prolongation of which 6 required transient cessation of ATO. Seventeen (13.8%) patients developed symptomatic sensory neuropathy requiring treatment. None required permanent discontinuation of therapy. At a median follow up of 854 days, there were 2 (1.6%) hematologic relapses and no deaths beyond induction therapy. The 2-year OS and EFS for the cohort (n=123) are 93.4% ± 2.3% and 91.6% ± 2.6% respectively (Figure 1c). Two (1.6%) patients have developed second malignancies. In a real world setting, concurrently administered ATO and ATRA with minimal anthracycline use results in excellent long term survival, even in the high risk group. Early deaths due to delayed presentation with infections and life threatening bleeding remain an unmet need for future research along with the need for strategies to reduce treatment-related toxicities. Figure 1 Figure 1. Disclosures Mathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.

Introduction: Blood coagulation analysis is routinely performed to assess bleeding and thrombotic risks in surgical and critical care patients as well as in patients with diseases that cause coagulation abnormalities (e.g., hemophilia, thrombophilia and sickle cell disease). Majority of coagulation assays are based on photo-optical measurement of coagulation onset in blood plasma such as prothrombin time (PT), international normalized ratio (INR), and activated partial thromboplastin time (aPTT) and viscoelastic measurement of coagulating whole blood, often referred to as "global coagulation analysis", mostly done by thromboelastography (TEG, ROTEM) but they require large sample volume (> 0.5ml) requiring venipuncture, have poor standardization, and are unreliable tools to predict bleeding/thrombotic risk. Acoustic tweezing coagulometry (ATC) is an innovative noncontact drop-of-blood coagulation analysis technique that can perform both photo-optical and viscoelastic coagulation analysis with a sample volume as low as 4 μl to provide a comprehensive set of clinically relevant coagulation parameters such as blood viscosity, elasticity, reaction time, clotting rate, maximum clot stiffness, fibrin formation rate and cross-linking kinetics helpful for diagnosis and prediction of bleeding and thrombotic risks. ATC is particularly valuable for the pediatric patients as it enables safe and reliable point of care coagulation assessment with minimal sample volume. Materials and Methods: In this project, we demonstrate the feasibility of ATC for coagulation analysis by validation and standardization of the technique using whole blood collected from healthy adult volunteers and commercially purchased blood plasma. Further, we present the ability of ATC to assess bleeding risk in commercial blood plasma with coagulation FVIII deficiency with and without inhibitors, as well as whole blood collected from pediatric Hemophilia A patients without inhibitors. The time dependent changes in elasticity (elastic tweezograph, Figure 1A) and viscosity (viscous tweezograph, Figure 1B) of coagulating blood plasma or whole blood sample are used to extract the following coagulation parameters: clot initiation time (CIT), clotting rate (CR), clotting time (CT), time to firm clot formation (TFCF), and maximum clot stiffness (MCS) from elastic tweezograph; reaction time (RT), fibrin formation rate (FFR), and maximum fibrin level (MFL) from viscous tweezograph. Results and Discussion: Figure 1C shows the elastic tweezograph and figure 1D shows the viscous tweezograph of the healthy plasma, plasma with coagulation FVIII deficieny and plasma with inhibitors for coagulation FVIII activated via the intrinsic pathway of coagulation. The tweezographs suggest that the clot initiation is faster in healthy plasma compared to the FVIII deficient plasma and FVIII inhibitor plasma. The clotting rate is highest for healthy plasma followed by the FVIII deficient plasma and is the lowest for the plasma with FVIII inhibitors suggesting a delayed clot formation in the deficient and inhibitor groups. They all reach a similar final clot stiffness, but the time to firm clot formation is least in healthy plasma as expected and increases in the FVIII deficient group and further increases in the FVIII inhibitor group. Conclusions: Acoustic tweezing coagulometry can successfully measure the viscosity, elasticity and coagulation of whole blood and blood plasma with only a drop of the sample. This technique can successfully assess the bleeding risks in pediatric and adult patients with Hemophilia. Acknowledgements: This study has been supported by American Heart Association pre doctoral fellowship 20PRE35210991, U.S. National Science Foundation grant 1438537, American Heart Association Grant-in-Aid 13GRNT17200013, and Tulane University intramural grants. The acoustic tweezing technology is protected by pending patents PCT/US14/55559, PCT/US2018/014879 and PCT/US21/15336. Figure 1 Figure 1. Disclosures Kasireddy: Levisonics Inc.: Current Employment. Rafique: Pfizer Inc.: Consultancy; CSL Behring: Consultancy; HEMA Biologics: Consultancy. Khismatullin: Levisonics Inc.: Current equity holder in publicly-traded company; Levisonics Inc.: Patents & Royalties: PCT/US14/55559 (pending); Levisonics Inc.: Patents & Royalties: PCT/US2018/014879 (issued) ; Levisonics Inc.: Patents & Royalties: PCT/US21/15336 (pending)..

Bendamustine was approved by the United States Food and Drug Administration in 2008 for the treatment of Chronic Lymphocytic Leukemia (CLL). Corticosteroids have been administered in regimens with chemotherapy for CLL for more than 40 years. Despite this, the combination of Bendamustine and Methylprednisolone has not been explored in CLL. We describe our experience with Bendamustine 100 mg/m2 and Methylprednisolone 125 mg IV on days 1 and 2 and Peg-filgastrim 6 mg subcutaneously on day 3 every 28 days for 3-4 cycles in 12 patients with CLL. Cycles have been limited to attempt to prevent cumulative marrow toxicity due to Bendamustine. Patient characteristics: 7 males/5 females, median age-58, Binet stage B (8), C (2), A (2); ZAP70/CD38+ (5), Trisomy 12 (3), 13q- (2), normal cytogenetics (2); median ECOG status 1, no prior therapy in 8. Median number of cycles received = 3 (two received 4 cycles). Most common toxicities: nausea/emesis (5), fever/flu-like symptoms (4), arthralgia/myalgia (4). There have been no treatment-related deaths. One patient was briefly hospitalized for pneumonia. Complete responses have been seen in 6 patients (median duration = 50.7 months; range: 12.1+ to 87.3 months). Partial responses have been seen in 4 patients (median duration = 17.3+ months; range: 3.3 to 26.9+ months). The combination of Bendamustine and Methylprednisolone given in a limited schedule is well-tolerated and is active in CLL. Disclosures No relevant conflicts of interest to declare.

In our earlier work with arsenic trioxide (ATO) resistance in acute promyelocytic leukemia (APL), we observed that ATO resistant cells reprogrammed their metabolism from glycolysis to oxidative phosphorylation (OXPHOS) as a mechanism of resistance. We further demonstrated that it could be overcome by targeting this metabolic switch using FCCP (mitocan) in combination with ATO (Balasundaram N et al. Biorxiv 2020). There is increasing evidence that acute myeloid leukemia (AML) cells have a greater metabolic plasticity unlike ATO resistant APL cells and most cancers that rely on glycolysis. AML leukemic stem cells preferentially utilize OXPHOS for their survival (Lagadinou ED et al. Cell stem cell 2013). Mitocans like venetoclax used in combination with hypo-methylating agents are already well established in the management of AML (Pollyea D, et al. Nat Med 2021). ATO is also an effective glycolytic inhibitor (Zhang H, et al. PNAS 2015) hence we hypothesized that a combination of ATO and mitocans could potentially target the metabolic plasticity of AML cells. As the combination of ATO and FCCP was found to be non-specific we performed a small-scale screening on an AML cell line (U937) using FDA-approved compounds that are reported to target mitochondria (Gohil V et al. Nature Biotechnology, 2010). Though most of the mitocans showed predicted synergy with ATO. We focused on artesunate (ART) as a candidate for further evaluation due to its specificity for malignant cells, high therapeutic index, bioavailability, route of administration, cost-effectiveness, and global usage as an antimalarial. The combination of ATO+ART significantly reduced the viability of different subtypes of AML cell lines (THP-1, MV4:11, and Kasumi-1) and acute lymphoblastic leukemia cell lines (Jurkat E6.1, SUP B15, and MOLT-4) with minimal effect on the normal cells (CD34 and peripheral blood mononuclear cells; n=10; 48hours) (figure 1a). We noted that the selective specificity of the combination was primarily due to the iron metabolism of the leukemic cells and a requirement of iron for the activity of ART. When an iron chelator deferoxamine (DFO) was used in combination with ATO+ART there was a significant reduction in the activity of the combination on the AML cells (Figure 1b, U937; n=10; 48hours). Seahorse extracellular flux analysis validated that ART (5uM) as a single agent promoted uncoupled mitochondrial respiration and the addition of ATO resulted in a metabolic catastrophe (figure 1c and d). Chemical drug proteomic analysis using biotinylated artesunate and pull down from the leukemic cells revealed that the top interacting partners were localized in the mitochondria. We also noted that ART treatment significantly affected the mitochondrial dynamics of leukemic cells, where ART and ATO+ART treated cells had fragmented mitochondria in comparison to the control and ATO alone treated cells where the mitochondria were more elongated (figure 1e, U937 cells; n=3). We evaluated the effect of ATO+ART and their combination with azacytidine (triplet) in-vitro. Dual and triple combinations showed greater toxicity on AML cell lines and primary AML cells (Figure 1f, n=50) in comparison to the normal peripheral blood mononuclear cells (PBMNCs) and normal CD34+ cells. Taken together, these findings highlight the selective specificity of these combinations and its clinical potential in AML. Figure 1 Figure 1. Disclosures Augustin: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor. Krishna: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; KCM Vellore: Patents & Royalties; SGUL: Patents & Royalties; AML: Other: Co-Inventor. Mathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.

The mechanisms regulating the sequential steps of terminal erythroid differentiation remain largely undefined, yet are relevant to human anemias that are characterized by ineffective red cell production. Erythroid Kruppel-like Factor (EKLF/KLF1) is a master transcriptional regulator of erythropoiesis that is mutated in a subset of these anemias. Although EKLF's function during early erythropoiesis is well studied, its role during terminal differentiation has been difficult to functionally investigate due to the impaired expression of relevant cell surface markers in Eklf(-/-) erythroid cells. We have circumvented this problem by an innovative use of imaging flow cytometry to investigate the role of EKLF in vivo and have performed functional studies using an ex vivo culture system that enriches for terminally differentiating cells. We precisely define a previously undescribed block during late terminal differentiation at the orthochromatic erythroblast stage for Eklf(-/-) cells that proceed beyond the initial stall at the progenitor stage. These cells efficiently decrease cell size, condense their nucleus, and undergo nuclear polarization; however, they display a near absence of enucleation. These late-stage Eklf(-/-) cells continue to cycle due to low-level expression of p18 and p27, a new direct target of EKLF. Surprisingly, both cell cycle and enucleation deficits are rescued by epistatic reintroduction of either of these 2 EKLF target cell cycle inhibitors. We conclude that the cell cycle as regulated by EKLF during late stages of differentiation is inherently critical for enucleation of erythroid precursors, thereby demonstrating a direct functional relationship between cell cycle exit and nuclear expulsion.

Introduction Direct acting oral anticoagulants are rapidly replacing warfarin, the most commonly used anticoagulant for patients with atrial fibrillation, pulmonary embolism (PE) and deep vein thrombosis (DVT). However, there is limited data on the efficacy and adverse effects of the commercial Kcentra used in these patients. Kcentra, the only approved PCC in the USA, is FDA approved for urgent reversal of acquired coagulation factor deficiency induced by vitamin K antagonist (VKA) therapy in adult patients with acute major bleeding or need for an urgent surgery/invasive procedure. In this study we examine the indications for administration of Kcentra, efficacy of the PCC in several coagulopathies, incidence of adverse events including ischemic stroke and DVT/ PE, and overall mortality. Methods Retrospective analysis of inpatients at UPMC Presbyterian, Shadyside and Mercy, admitted between February 2016 and 2017 was done. A total of 213 patients, with history of stroke / DVT / PE who received Kcentra were grouped based on the cause of coagulopathy, sites of bleed (CNS or non-CNS) or the clinical setting of Kcentra administration. Definitions of the International Society on Thrombosis and Hemostasis (ISTH) / Scientific and Standardization Committee were used to assess the efficacy of management. Among the patients on Warfarin, only non-CNS and prophylactic administration group were included here as those with CNS bleeding were part of an earlier study. Adverse events measured were incidence of ischemic stroke and DVT/ PE during their hospital stay. Results The 213 patients who received Kcentra included those on warfarin (40%), apixaban (21%), Rivaroxaban (23%), with the remaining 16% including patients with hepatic cirrhosis, disseminated intravascular coagulation and those with coagulopathy related to heparin/ fondaparinux or antiplatelets (ASA, clopidogrel). The study group included those with CNS bleeds (33%), non-CNS bleeds (37%) and those in the prophylactic, pre-operative setting (30%). The majority of the non-CNS bleeds were patients with gastrointestinal bleeding (45%), with the rest including the musculoskeletal (31%) and intra-abdominal / intra-thoracic (16%) bleeding. Mortality was highest in patients with cirrhosis (n = 15), DIC (n = 8), and on anti-platelet medications (n = 2) at 100%, followed by 60% in patients on heparin / fondaparinux (n = 5). The mortality was markedly lower (~13%) in patients on Coumadin, Apixaban and Rivaroxaban. 2 of the patients, previously on rivaroxaban, developed multiple CNS infarcts. Thromboembolic events were significantly higher (40%) in patients with cirrhosis, with moderate effect in the Rivaroxaban group (14.5%) when compared to coumadin (2.3%) and apixaban (0%). Conclusions Kcentra was used in several off-label clinical settings, with comparable mortality among the coumadin, rivaroxaban and apixaban groups and no identifiable benefit in the setting of cirrhosis, DIC or antiplatelet medications, but with an increased incidence of deep vein thrombosis and stroke. Disclosures No relevant conflicts of interest to declare.

Introduction: Measurement and interpretation of mechanical properties of whole blood and blood plasma are important diagnosis and treatment monitoring of various conditions like coagulopathy, hemophilia, sickle cell disease and many cardiovascular disorders. Many of the current techniques like thromboelastography, micro-viscometry or microfluidic devices used for this purpose require a large sample volume and/or may be prone to measurement errors due to sample contact with device walls. To address these issues, we developed a single-drop non-contact method for blood rheological analysis, referred to as "acoustic tweezing rheometry". With sample volume as small as 4 μL, our innovative technology has been successfully applied for assessment of whole blood and blood plasma coagulation. Here, we present the extension of this technology to resonant spectroscopic measurement of blood viscoelasticity. Materials and Methods: The schematic of the acoustic tweezing device is shown in (Figure 1A). The standing acoustic wave field between the transducer and reflector generates the acoustic radiation force on the biological sample that traps it in a host fluid (e.g., air). Sample tweezing (force-induced deformation and translational motion of the trapped sample) is achieved by amplitude modulation of the acoustic tweezing signal at high frequency and then decrease the frequency continuously until the lower limit for sample trapping is reached. During this frequency sweep, shape changes of the sample were recorded (Figure 1B) by a photodetector and a high-speed camera (Figure 1A). The amplitude-frequency response of the sample was obtained from raw data analysis, with the amplitude being the maximum deflection of the sample height from its equilibrium value. Dynamic (shear) viscosity and elasticity of the sample were assessed from the quality factor of the amplitude-frequency response (Figure 1C) and the resonance frequency, respectively. Results and Discussion: The quality factor analysis predicted that the dynamic viscosity of commercial normal control blood plasma was 1.5 mPa·s at room temperature, which agreed with previous large-sample-volume measurements. Once re-calcified, the resonance frequency of blood plasma and thus its shear elasticity increased due to clot formation until reaching a plateau in 5 min (Figure 1D). Using this graphical output (referred to as "tweezograph"), the following coagulation parameters can be extracted: clot initiation time, clotting rate, clotting time, and maximum clot elasticity. Conclusions: Resonant acoustic tweezing spectroscopy can accurately measure dynamic viscosity and elasticity of whole blood and blood plasma with a small drop of the sample and without artefacts or measurement errors due to sample contact with device walls. This technique can be applied for rapid assessment of whole blood and blood plasma coagulation. Acknowledgments: This study has been supported by U.S. National Science Foundation grant 1438537, American Heart Association Grant-in-Aid 13GRNT17200013, and Tulane University intramural grants. The acoustic tweezing technology is protected by pending patents PCT/US14/55559 and PCT/US2018/014879. Disclosures Khismatullin: Levisonics Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Background: New prognostic factors have been recently identified in AML patient population that include frequent mutations of receptor tyrosine kinases (RTK) including KIT, PDGFR, FLT3, that are associated with higher risk of relapse. Thus, targeting RTKs could improve the therapeutic outcome in AML patients. Aim: To create a digital drug model for dasatinib and validate the predicted response in AML patient samples with ex vivo drug sensitivity testing. Methods: The Beat AML project (supported by the Leukemia & Lymphoma Society) collects clinical data and bone marrow specimens from AML patients. Bone marrow samples are analyzed by conventional cytogenetics, whole-exome sequencing, RNA-seq, and an ex vivo drug sensitivity assay. For 50 randomly chosen patients, every available genomic abnormality was inputted into a computational biology program (Cell Works Group Inc.) that uses PubMed and other online resources to generate patient-specific protein network maps of activated and inactivated pathways. Digital drug simulations with dasatinib were conducted by quantitatively measuring drug effect on a composite AML disease inhibition score (DIS) (i.e., cell proliferation, viability, and apoptosis). Drug response was determined based on a DIS threshold reduction of > 65%. Computational predictions of drug response were compared to dasatinib IC50 values from the Beat AML ex vivo testing. Results: 23/50 (46%) AML patients had somatic mutations in an RTK gene (KIT, PDGFR, FLT3 (ITD (n=15) & TKD (n=4)), while 27/50 (54%) were wild type (WT) for the RTK genes. Dasatinib showed ex vivo cytotoxicity in 9/50 (18%) AML patients and was predicted by CBM to remit AML in 9/50 AML patients with 4 true responders and 5 false positive. Ex vivo dasatinib responses were correctly matched to the CBM prediction in 40/50 (80%) of patients (Table1), with 10 mismatches due to lack of sufficient genomic information resulting in profile creation issues and absence of sensitive loops in the profile. Only 4/23 (17%) RTK-mutant patients and 5/27(19%) RTK-WT patients were sensitive to dasatinib ex vivo, indicating that presence of somatic RTK gene mutations may not be essential for leukemia regression in response to dasatinib. Co-occurrence of mutations in NRAS, KRAS and NF1 seemed to associate with resistance as seen in 10 of the 14 profiles harboring these mutations. Conclusion: Computational biology modeling can be used to simulate dasatinib drug response in AML with high accuracy to ex vivo chemosensitivity. DNA mutations in RTK genes may not be required for dasatinib response in AML. Co-occurrence of NRAS, KRAS and NF1gene mutations may be important co-factors in modulating response to dasatinib. Disclosures Tyner: Leap Oncology: Equity Ownership; Syros: Research Funding; Seattle Genetics: Research Funding; Janssen: Research Funding; Incyte: Research Funding; Gilead: Research Funding; Genentech: Research Funding; AstraZeneca: Research Funding; Aptose: Research Funding; Takeda: Research Funding; Agios: Research Funding. Druker:Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Research Funding; Millipore: Patents & Royalties; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; McGraw Hill: Patents & Royalties; Celgene: Consultancy; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Patient True Talk: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Fred Hutchinson Cancer Research Center: Research Funding; Beta Cat: Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy. Sahu:Cellworks Research India Private Limited: Employment. Vidva:Cellworks Research India Private Limited: Employment. Kapoor:Cellworks Research India Private Limited: Employment. Azam:Cellworks Research India Private Limited: Employment. Kumar:Cellworks Research India Private Limited: Employment. Chickdipatti:Cellworks Research India Private Limited: Employment. Raveendaran:Cellworks Research India Private Limited: Employment. Gopi:Cellworks Research India Private Limited: Employment. Abbasi:Cell Works Group Inc.: Employment. Vali:Cell Works Group Inc.: Employment. Cogle:Celgene: Other: Steering Committee Member of Connect MDS/AML Registry.

Introduction For chimeric antigen receptor T cell-based (CAR-T) and engineered T cell receptor (TCR) immunotherapies, T cell expansion methods and phenotype/s of transplanted T cells may heavily influence clinical outcomes. Much current focus is on the potential of defined CD4+/CD8+ T cell populations vs bulk, and on the potential superiority of CAR-T cells from naïve (TN) or central memory (TCM) versus effector memory (TEM) cells. Many commercial T cell activation and expansion methods utilize rigid magnetic beads bound to antibodies against CD3 and CD28 as substrates. These methods are often associated with high costs and licensing restrictions for clinical and commercial applications. Additionally, de-beading processes can be highly complex and inefficient, adding additional time, costs and risks. It has been shown that substrate rigidity influences T cell expansion and phenotype. We hypothesized that a novel phase-change substrate could modulate expanded T cell phenotype/s and address de-beading challenges. Methods An alginate-based phase-change hydrogel was synthesized and coated onto magnetic beads to form hydrogel-coated particles of approximately 10 µm diameter. This hydrogel, in the presence of chelating agents, rapidly dissolves, enabling removal magnetic bead removal. The coated particles were conjugated with streptavidin (SA) and bound to biotinylated antibodies against CD3 (OKT3) and CD28 (28.2) to form CD3/CD28 hydrogel particles (CD3/CD28-HP). Human CD3+ T cells from peripheral blood were seeded (Day 0) at 1x10E6 cells/mL in 24 well plates (n=3) in complete RPMI medium supplemented with IL-2. To each well, 25 µL of CD3/CD28-HP were added per 0.5x10E6 cells in a single stimulation. Media addition or change of culture vessel occurred each 2-3 days. Following expansion, chelating agent was added and magnetic beads removed. Flow cytometry was used to assess cell viability and expression of phenotypic markers including CD3, CD4, CD8, CD45RA and CCR7. ELISA was used to measure secretion of IL-2, IL-4, and IFNγ. Residual magnetic beads were counted via hemocytometer. Results CD3/CD28-HP promoted significant T cell expansion of 0.3, 1.4, 2.4, 4.8 and 6.6 population doublings (PD) by Days 2, 5, 6, 9, and 13 respectively (p Phenotypic markers were assessed on Days 6 and 13. Expansion using CD3/CD28-HP led to significantly more CD8+ cells and significantly fewer CD4+ cells versus the starting population on both days (p Following de-beading of expanded cells, cell recovery was 96% for the CD3/CD28-HP-expanded cells and 93% for cells expanded using commercial magnetic bead-based expansion product. Additionally, in de-beaded cells, fewer residual magnetic particles were present in the CD3/CD28-HP-expanded population than in cells expanded via the commercial magnetic bead-based expansion product. Conclusions These data demonstrate the utility of a novel phase-change hydrogel system to efficiently induce T cell proliferation, promote expansion of functional T cells expressing markers associated with CD8+, TN and TCM phenotypes, and to separate expanded cells efficiently from magnetic beads. In future studies, we will determine if T cells expanded using this method show increased stemness and persistence in in vivo models, and further explore the possibilities of this novel system for rapid expansion and recovery of specific T cell subtypes. Disclosures Jesuraj: Quad Technologies: Employment, Other: stock options. Cole:Quad Technologies: Employment, Other: Stock Options. Wells:Quad Technologies: Employment, Other: Stock Options. Qin:Quad Technologies: Employment, Other: Stock options. Kevlahan:Quad Technologies: Employment, Equity Ownership. Maus:Novartis: Patents & Royalties: related to CTL019, Research Funding. Ball:Quad Technologies: Employment, Other: Stock Options.

There are numerous reports on bone marrow micro-environment mediated drug resistance (EM-DR) in cancer. We had previously reported, that there was significant EM-DR to ATO in APL. We had also shown that the effect of cross talk between leukemic cells and stromal cells leads to increased activation of NF-kB pathway in the leukemic cells. There are very limited data available on the effect of leukemic cell interaction on the stromal cells. We undertook a study to evaluate the changes induced by leukemic cells (NB4 cell line) on the HS-5 stromal cell line. After 2 days of co-culture with NB4 cells, the RNA from HS-5 stromal cells were subjected to gene expression profiling and small RNA sequencing. In a gene expression profiling, we observed an enrichment of Wnt signaling and BMP signaling pathway. When we subjected the array to functional analysis, a significant enrichment of osteoblast differentiation genes was observed in the stromal cells co-cultured with leukemic cells in comparison to HS-5 cells alone. A stringent miRNA analysis using DESeq (miRNAs whose FDR corrected p values < 0.05 are considered significant) revealed 21 miRNA were differentially regulated in the HS-5 cells co-cultured with leukemic cells in comparison to HS-5 cells alone. Consistent with the gene expression profile, we observed that most of the miRNA differentially regulated were involved in the regulation of osteoblast differentiation (such as hsa-miR-33b-5p, hsa-miR-210-3p and hsa-miR-222-5p). As previously reported (PNAS 2013;110 (23):9469) we also observed upon co-culture with leukemic cells a down-regulation of NF-kB pathway in HS-5 cells. This was demonstrated by documenting decreased translocation of p65 subunit in the nuclear compartment of HS-5 cells on day 2 and day 7 of co-culture. This was further validated by documenting downregulation of NF-kB pathway genes by real time PCR on day 2. Along with this we also observed an increased translocation of B-catenin in the nuclear compartment of HS-5 cells on day 2 and day 7 of co-culture. This observation was validated by a real time PCR assay where an upregulation of its target gene was seen (Fig 1a). We observed that there is an increased expression of osteoblast differentiation transcription factor (RUNX2) in HS-5 cells upon co-culture on day 2 by real time PCR and western blot (Fig 1a, 1b). We also observed an increase in alkaline phosphatase activity in HS-5 cells upon co-culture with NB4 cells at different time points detected by BCIP-NBT staining. Further validation of differentiation of HS-5 cells into osteoblast was done using alizarin red S stain on day 7. Similar differentiation effects were seen in HS-5 cells when it is co-cultured with other malignant cells (U937, U266 and Jurkat) where an increased expression of RUNX2 on day 2 of co-culture was observed, suggesting that this effect is common with different subtypes of leukemia. We did not observe any effect of PBMNC / normal cord blood CD34+cells on HS-5 cells in inducing osteoblast differentiation (no expression of RUNX2; Fig 1b). This study demonstrates a possible role of leukemic cells in establishing leukemic niche in the bone marrow by inducing osteoblast differentiation of stromal cells. The role of this leukemic cell induced osteoblast differentiation of stroma in drug resistance against various chemotherapeutic agents needs further evaluation. Disclosures No relevant conflicts of interest to declare.

Recent evidences suggests that the efficacy of Lenalidomide (LEN) depends upon its ability to degrade IKZF1 and IKZF3 proteins via cereblon dependent ubiquitin proteasome pathway [Science. 2014 Jan 17; 343(6168): 301-305]. Based on this model it would theoretically be antagonistic to combine LEN with proteasome inhibitors (PI). However, it is well recognized that there is significant synergism when LEN is combined with PI and this combination is routinely and effectively used in the clinic. The mechanism of synergy and the fate of IKZF1 and IKZF3 when these two agents are combined is poorly understood. We undertook a series of experiments to study the fate of IKZF1 when this combination of drugs was used in multiple myeloma cells. Combining LEN (1uM) along with bortezomib (BTZ; 1nM) a PI showed a significant kill on U266 cells (myeloma cell line) on day 5 post treatment (n=3; P=0.02) when compared to either of the agents alone. In an MTT assay, the synergism was well documented with a combination index of 0.5 (Fig 1a). Next we assessed the function of proteasome (chymotrypsin activity) when LEN was combined with PI. We observed that LEN alone does not interfere with proteasome activity. It was noted that BTZ alone at the concentration used (5 nM) was able to effectively inhibit the activity of proteasome (Fig 1b). It was also observed that combining these two agents does not interfere with BTZ action in inhibiting proteasome complex (Fig 1b). As a result of efficient proteasome inhibition, we observed an accumulation of ubiquitinated proteins in the BTZ and LEN + BTZ treated cells when compared to control and LEN alone treated cells (Fig 1c). Next, we looked for the fate of IKZF1 in U266 cells treated with LEN, BTZ and in combination of both the drugs. As reported, we observed a degradation of IKZF1 in U266 cells upon treatment with LEN. While we did not see any degradation of IKZF1 in BTZ alone treated cells. It was noted that in combination treated cells (LEN+BTZ) there was a degradation of IKZF1 (Fig 1c). In spite of significant proteasome complex inhibition, degradation of IKZF1 was observed which suggested a proteasome independent mechanism. It is well known that proteasome inhibition results in upregulation of the autophagy pathway which in turn can degrade the accumulated ubiquitinated proteins. We noted that upon treatment with BTZ or LEN+BTZ an induction of autophagy was observed, as evidenced by an increase in generation of LC3II bands on an immunoblot (Fig 1c). To support our hypothesis that IKZF1 is degraded by autophagy in the absence of proteasome complex, we pre-treated the U266 cells with an autophagy inhibitor (3-methyladenine) followed by treatment with LEN and BTZ and noted an accumulation of IKZF1 proteins (Fig 1d). We also observed a downregulation of IKZF1 target genes IRF4 and c-MYC by 12 and 24 hours in the combination treated cells (data not shown). Taken together this data demonstrates that there is (i) significant in-vitro synergism between the two agents (ii) the combination additively induces autophagy pathway (iii) IKZF1 protein can be degraded via this autophagy pathway in the presence of effective proteasome inhibition. While additional mechanisms of synergy between these two agents cannot be excluded, further enhancing autophagy pathway in these cells by drugs like sirolimus (autophagy inducer in myeloma cells) could potentially improve the synergy between these two drugs. Disclosures No relevant conflicts of interest to declare.

The focus of ATO resistance in acute promyelocytic leukemia (APL) has centered on mutations in PML-RARA gene (Blood 2011, NEJM 2014). However such mutations are rare and cannot explain the majority of relapses seen in the clinic. To evaluate the mechanisms of ATO resistance, we generated ATO resistant NB4 sub clone NB4-EVAsR1 (A216V - VAF-91.7%) in our laboratory. We also had another ATO resistant cell line (UF1) which does not have the A216V mutation. In an expression array we noted that redox signaling, AMPK signaling and energy metabolism pathways were significantly dysregulated in the ATO resistant cell lines compared to naïve NB4 cells. Towards validating the microarray data and to characterize the ATO resistant cell lines we measured the basal levels of reactive oxygen species (ROS), glutathione(GSH), mitochondrial membrane potential (MMP), glucose uptake and their sensitivity to glycolytic inhibitor 2-Deoxy glucose (2-DG) in comparison to naïve NB4 cells. We observed that resistant cell lines have significantly lower ROS, MMP, glucose uptake (Fig 1a) and increased GSH. We also observed that the resistant cell lines were significantly less susceptible to treatment with 2-DG in comparison to naïve NB4 cells (Fig 1b) suggesting that resistant cell lines were less dependent on glycolysis. ATO has been reported to directly inhibit the glycolytic pathway, this effect is believed to contribute to its cytotoxic effect (PNAS 2015). However, we did not observe any cytotoxic synergy between ATO and 2-DG on naïve NB4 cells and neither did this combination restore sensitivity to ATO in the resistant cell lines (Fig 1b). Next we assessed the sensitivity of these resistant cell lines to oxidative phosphorylation (OXPHOS) inhibitors. We used an uncoupler (FCCP at 10uM) of OXPHOS which promotes uncoupled respiration by deregulating the proton gradient which drives ATP synthesis via ATP synthase. We observed that the FCCP treatment alone did not reduced the viability of naïve NB4 cells. Similarly, viability of ATO resistant cell lines also did not reduce significantly suggesting the ability of these cells to uncouple their metabolic pathway from OXPHOS to glycolysis when inhibited. However, when FCCP was combined with ATO it significantly restored the sensitivity of the resistant cell lines to ATO (Fig 1c). The same combination did not have any additive effect on naïve NB4 cells. The combination not only restored the sensitivity of the ATO resistant cell lines but also sensitized the conventionally ATO resistant cell lines such U937 (Fig 1c) and THP1. In spite of the profound effect on leukemic cells we also observed a significant bystander effect on the normal peripheral blood mononuclear cells (Fig 1c). The data suggests that the sensitivity of these resistant cell lines could be potentially restored by combining ATO with an OXPHOS uncoupler. A number of molecules that are FDA approved and used in the clinic also have OXPHOS uncoupling activity and could potentially be evaluated for their synergistic activity with ATO in leukemia. This data also draws attention to possible severe systemic off-target toxicity of such combinations which may be inadvertently used in the clinic. Disclosures No relevant conflicts of interest to declare.

Abstract 825 Epigenetics has emerged as a key regulator of the fetal to adult hemoglobin switch during development. Understanding the mechanisms of fetal 𝛄-globin gene silencing offers the promise of effective targeted therapy of β- thalassemia and sickle cell anemia. Previous studies carried out by our group using adult erythroid cells from mice transgenic for a yeast artificial chromosome containing the entire human β-globin locus (β-YAC) and primary human erythroid cells have shown that methyl-CpG binding protein 2 (MBD2) is critical for full silencing of the fetal 𝛄-globin gene. MBD2 binds to methylated CpG rich promoters and silences the associated target genes by recruiting the Nuclear Remodeling and Deacetylase (NuRD) co-repressor complex. Absent or ≥75% decreased expression of MBD2 has been shown to have a 10–50 fold stimulatory effect on the expression of the 𝛄-globin gene in adult β-YAC transgenic mice and a 5–6 fold effect in CD34+ progenitor derived human erythroid cells in culture. Mi2, a major component of the NuRD complex, is an ATP-dependent chromatin remodeler consisting of two isoforms, Mi2a (also known as CHD3) and Mi2β (also known as CHD4). We have observed that 80% knockdown of Mi2β leads to a significantly higher expression of the 𝛄-globin gene in βYAC containing murine adult hematopoietic CID cells (∼100 fold) when compared to equivalent knockdown of other components of the MBD2/NuRD complex (∼5-10 fold), including MBD2. Remarkably, in CD34+ progenitor derived adult human erythroid cells, as little as 40% knockdown of Mi2β resulted in a ∼30 fold increase in 𝛄/𝛄 + β mRNA levels. Moreover, simultaneous knockdown of MBD2 and Mi2β resulted in no greater 𝛄-globin gene expression than Mi2β knockdown alone. This suggests that Mi2β is acting independently of as well as through its role in the MBD2-NuRD complex to exert its silencing effect on the 𝛄-globin gene. Complete conditional knockout of Mi2β in transgenic mouse hematopoietic cells has been shown by others to result in arrest of erythroid differentiation. We observed that in human CD34+ progenitor cells ≥80% Mi2β knockdown also altered differentiation. In contrast, 40–50% knockdown of Mi2β does not affect erythroid differentiation compared to scramble shRNA controls and resulted in 25 – 40% 𝛄/𝛄 + β globin RNA expression versus 2–3% in scramble shRNA controls. To determine possible mechanisms of Mi2β-mediated silencing outside of its role in the MBD2-NuRD complex, we examined the effect of Mi2β knockdown on two key mediators of 𝛄-globin gene silencing, Bcl11A and KLF-1 (EKLF). Our results showed that RNA and protein levels of these proteins are diminished by 40% and 70% respectively in cells with 40–50% Mi2β knockdown. Knockdown of MBD2 caused no decrease in either Bcl11A or KLF-1 levels. Thus at least part of the effect of Mi2β on 𝛄-globin gene silencing is through its positive effects on KLF-1 and Bcl11A expression. In summary, we have shown that less than a 50% decrease in Mi2β expression in primary human adult erythroid cells resulted in high levels of 𝛄/𝛄 + β gene expression without altering erythroid differentiation. The silencing effects of Mi2β occur both through the MBD2-NuRD repressor complex and through positive regulation of the KLF-1 and Bcl11A genes. These results also suggest that partial inhibition of Mi2β function could be of therapeutic benefit in the treatment of β- thalassemia and sickle cell anemia. Disclosures: No relevant conflicts of interest to declare.

Degradation of PML-RARA upon treatment with ATO is predominantly mediated by the proteasome complex. Reports suggest that in relapsed APL patients who were treated upfront with ATO, mutations in the B2 domain of PML in PML-RARA gene are involved in resistance to ATO. These mutations predict a poor clinical outcome in spite of subsequent combination of ATO with chemotherapy (NEJM 2014). We had previously reported that Bortezomib (Bo) was able to synergize with ATO by inducing apoptosis through increased levels of ROS and up regulation of the UPR pathway (Blood. 2012;120, 3552) and we had also noted PML-RARA degradation and nuclear body formation with this combination. We further evaluated the mechanism of degradation of PML-RARA when ATO was combined with Bo and the effect of this combination on resistant cell lines, a mouse model and in relapsed patients. We generated in-house ATO resistant NB4 cell lines (NB4EV-ASR1, ASR2 and ASR3). In NB4EV-ASR1 we confirmed the presence of A216V mutation in the PML B2 domain (previously reported to be involved in ATO resistance) while the other 2 clones did not have this or any other mutation in PML-RARA. We noted an increase in the baseline proteasomal activity in all the resistant cell lines when compared to naïve NB4 cells (n=3; data not shown). The combination of ATO and Bo induced a significant apoptosis in all the resistant cells similar to naïve NB4 cells (Figure 1A: n=4; Combination Index = 0.02).The mechanism of inducing apoptosis in the resistant cell lines was similar to naïve NB4 cells, as previously reported by us, and involved an increased level of ROS, decreased mitochondrial membrane potential, induction of UPR and activation of caspase-3 (Figure 1B). We next evaluated PML-RARA degradation in NB4 naive cells treated with a combination of ATO+Bo. At 24 hours, there was an evidence of induction in autophagy as shown by LC3II formation using western blot technique which increased at 48 hours; this time point coincides with time at which maximum PML-RARA degradation occurred (Figure1C). Similar results were seen in the resistant cell lines (with and without mutation A216V). Blocking autophagy by 3-methyl adenine showed a partial inhibition in the degradation of PML-RARA. We have also observed that there is an accumulation of p62 (ubiquitin binding protein) at 24 hours and this was degraded by 48 hours suggests that accumulated ubiquitinated products were cleared by autophagy via p62 (Figure1D). In a co-immunoprecipitation experiment, p62 and LC3II proteins precipitated along with PML-RARA (figure 1E). Knock down of p62 transcript by siRNA followed by ATO+Bo treatment showed an accumulation of PML-RARA in the treated cells in comparison to the scrambled and control. In an APL transplanted mice model, combination of ATO and Bo prolonged the life span of the mice as illustrated in Figure1F. In this group there was a significant decrease in the leukemia burden evidenced by decreased leukemic cells in bone marrow, peripheral blood and spleen by flow cytometry, RQ-PCR and decreased spleen size on day +20. A reduction in the LIC was demonstrated by secondary transplants. We also observed that transplantation of bone marrow cells from the long term surviving mice post ATO+Bo therapy did not induce leukemia (Figure1G) and no transcripts of PML-RARA were detected in the recipients. A phase II clinical study combining Bo with ATO and chemotherapy has been initiated for patients with relapsed APL (NCT01950611). In this ongoing study 11 patients have been enrolled. The median age was 32 years. 7 were males. All patients achieved hematological remission and the median time to complete molecular remission was 42 days (one patient still on induction therapy). The addition of Bo was well tolerated. None of the cases had evidence of significant neuropathy, worsening of coagulopathy, IC bleed or a differentiation syndrome. Long term follow up is awaited to comment on the efficacy of this combination. In conclusion, the mechanism of ATO+Bo synergy is multi-factorial and appears to be predominantly due to increase in ROS activity and up regulation of UPR pathway leading to apoptosis. In spite of proteasomal inhibition by addition of Bo with ATO, PML-RARA continues to be degraded and this is mediated by up-regulation of autophagy pathway. ATO+Bo synergy was further confirmed in a pre-clinical model. This combination is also effective in ATO resistant cell lines with high levels of synergism. Figure 1 Figure 1. Disclosures Off Label Use: Bortezomib in the treatment of acute promyelocytic leukemia.

There has been a recent concern of arsenic trioxide (ATO) resistance in patients with acute promyelocytic leukemia (APL) treated with ATO as upfront therapy. The focus of ATO resistance has centred on mutations in PML-RARA gene (Blood 2011, NEJM 2014). NB4 cells in our laboratory were exposed to serial increasing concentrations of ATO. We subsequently generated 3 ATO resistant clones, NB4EV-ASR1, ASR2 and ASR3. In addition we have also evaluated an established ATRA resistant APL cell line UF1 (Gift from Dr. Chomienne. C). In a viability assay, we observed that these cell lines are resistant to ATO and had an IC50 above 2µm (Table1). These resistant cell lines also had a higher IC50 to other therapeutic drugs such as Daunorubicin and Cytosine arabinoside and a reduced differentiation effect on exposure to ATRA (summarized in Table 1). To identify the differences between the naïve cells and the resistant cells, we did whole exome sequencing (NGS) by Iontorrent and found that only NB4 EV-ASR1 clone had ATO resistance causing mutation (A216V) in the PML B2 domain (VAF=91.7%) while the other two cell lines (NB4EV-ASR2 and ASR3) did not have a mutation in PML-RARA. Next, we did an expression array to find the differential regulated genes between the naïve cell line and the parent resistant cell line from where all the 3 cell lines had been derived. We found that 1490 genes were differentially regulated (> 2 fold). The pathways significantly enriched for differentially expressed genes were cell survival, ABC transporters, Glutathione synthesis, Ubiquitin- proteasome degradation system and signalling pathways like PI3-AKT and PTEN. Validating the micro-array data, we found that there is an increased expression of ABC transporters such as MRP4, AQP9 (n=3; Figure1A) which are known to efflux ATO from the cells and a decreased expression of ABCA1 (known to efflux glutathione). The up regulation of these transporters also correlated with decreased levels of intracellular ATO (IC-ATO; measured using AAS, see Figure 1B for details) in the resistant cell lines. We also noted that there is a varying reduction in the basal reactive oxygen species levels and a varying increase in the amount of basal reduced glutathione (GSH) levels in the resistant cell lines (n=3, Table 1). We have noted that adding Buthionine sulphoximine (BSO - GSH inhibitor) along with ATO was able to restore the sensitivity of ATO in the resistant cells lines, however there was significant variation in the sensitivity of ATO among the cell lines when treated with the same concentration of BSO (Figure 1C). At the transcript levels we did not find any difference in expression of PML-RARA but at the protein level we noted a significant reduction in the levels of PML-RARA in the resistant cell lines (Figure 1D). We also observed an increase in the proteasome activity in the resistant cell lines compared to naïve cells (data not shown). In an immunofluorescence assay probing for PML, we found an absence of micro-speckled pattern in the resistant cell lines and UF1 cell lines compared to naive cells (Figure 1D). In conclusion, we have observed that in addition to PML-RARA mutations, variations in the Redox system, ABC transporters, intracellular ATO concentration and anti-apoptosis pathways are likely to be altered in ATO resistance. It is likely that ATO resistance is multi-factorial and that the dominant mechanism can vary between different resistant cell lines and potentially the same variation could be seen in relapsed patients. Importantly in the presence of ATO resistance there was also a decrease in sensitivity to other conventional agents used to treat APL. Novel agents and strategies based on these observations are required to address the issue of ATO resistance in patients with relapsed APL. Abstract 3554. Table. Characteristic features NB4 naïve NB4EV-AsR1 NB4EV-AsR2 NB4EV-AsR3 UF1 Sensitivity to ATO (IC 50 -µM) 0.9 3.09 3.44 2.88 4.1 Differentiation with ATRA exposure (1uM for 72hrs) (n=4) (CD11b% expression) (mean±SD) 49.2±7.3 40.1±3.0 12.4±2.5 30.5±2.6 0.57±0.23 Sensitivity to other chemotherapy drugs (IC50) (n=3) a) Daunorubicin(µM) b) Cytosine arabinoside (µM) 0.14 8.3 0.22 16.5 0.19 4.7 0.2 13.1 0.18 NA MRP4 expression (Fold difference) 1 4.2 3.8 4.2 NA Reactive oxygen species (ROS) levels (MFI Fold difference normalized to NB4 cells) (n=3) 1 0.74 0.86 0.68 0.3 Glutathionine levels measured by flowcytometry (MFI Fold difference normalized to NB4 cells) (n=3) 1 1.37 1.45 1.39 0.5 Disclosures No relevant conflicts of interest to declare.