ALBERT

Description: Background: Calreticulin (CALR) mutations are one of the major driver mutations in BCL-ABL1-negative myeloproliferative neoplasm (MPN) and are frequently detected in JAK2/MPL-unmutated essential thrombocythemia and primary myelofibrosis. Mutant CALR activates JAK-STAT signaling through an MPL-dependent mechanism to mediate pathogenic thrombopoiesis in MPNs. Although JAK inhibitors such as ruxolitinib can provide important clinical benefits to MPN patients including those harboring CALR mutations, JAK inhibition does not preferentially target the MPN clone and acquired resistance develops over time. We aimed to characterize the mechanisms of acquired resistance to JAK inhibitors in CALR-mutated hematopoietic cells and to screen for novel therapeutic approaches specifically target CALR-mutant cells in this study. Methods: UT-7/TPO-derived cell lines expressing wild-type and type 1 and type 2 mutant CALR (CALRdel52 and CALRins5) were kindly provided by Drs. Komatsu and Araki. JAK2-inhibitor-resistant cells were generated by co-cultured with ruxolitinib and fedratinib (TG101348, a JAK2-selective inhibitor). JAK-STAT signaling was evaluated by Western blot on CALR-wild-type and mutated cells exposed to JAK2 inhibitor compared to untreated cells. For the detection of acquired secondary mutations in CALR-mutated cells exposed to JAK2 inhibitor, whole exome sequencing (WES) was performed using the BGISEQ-500 Sequencing platform (BGI, Shenzhen, China) with the 2 x 100 bp paired-end protocol. Genome Analysis Toolkit was used for variation detection. Reads were aligned to human reference genome hg19 using BWA version 0.7.15. Targeted resequencing was performed on leukocytes from patients with MPN who had been treated with ruxolitinib. Screening with chemical libraries/novel compounds will be conducted on UT7/TPO-CALR cell lines. Results: Compared to the parental cells, ruxolitinib-resistant UT7/TPO-CALR mutant cell lines have developed significant cross resistance to other JAK inhibitor as shown in the cell viability study. Signalling downstream of JAK2 in all 3 inhibitor-naïve UT-7/TPO/CALR parental cell lines was inhibited by acute treatment of ruxolitinib as shown on Western blot. Whereas, constitutive JAK2 activation was observed in all 3 inhibitor-resistant UT-7/TPO/CALR cell lines. No change in the expression of Epo and MPL receptors in these cell lines was found. Interestingly, constitutive JAK3 activation was also seen in inhibitor-resistant UT-7/TPO/CALR cells in comparison with parental cells. These findings indicated the presence of transphosphorylation by JAK3 in inhibitor-resistant UT-7/TPO/CALR cell lines. In addition, the results of WES identified several acquired secondary mutations in 3 inhibitor-resistant UT-7/TPO/CALR cell lines including SH2B1, ABCC1, HOXB3 and KRTAP4-5. No acquired secondary mutation was identified in CALR and other genes involved in JAK-STAT signaling. Acquired secondary mutation will be screened in primary MPN patients' samples treated with JAK inhibitor. Type II JAK inhibitor such as BBT-594 has been shown to inhibit JAK activation and signaling in JAK-persistent/resistant cells. Conclusions: Our results confirmed that the in vitro efficacy of JAK2 inhibition on CALR-mutant cells. Our data also suggested that JAK2 transphosphorylation and acquired secondary mutations could be underlying mechanisms for acquired resistance to JAK inhibitors in CALR-mutated cells. Novel therapeutics approaches should be developed to overcome acquired resistance in CALR-mutated cells. Disclosures No relevant conflicts of interest to declare.

Description: Background: B cell maturation antigen (BCMA) is a potential therapeutic target in multiple myeloma. CT053 CAR-BCMA, which is a BCMA-specific chimeric antigen receptor (CAR) T cell, consists of autologous T cells genetically modified with a second-generation CAR incorporating a fully human anti-BCMA single chain fragment variant, a 4-1BB co-stimulatory domain and a CD3-zeta signaling domain. CT053 was studied in a single-arm, open-label phase I program (NCT03716856, NCT03302403, and NCT03380039) in eastern China. Methods: This investigation is a 3-site study in adult subjects with relapsed/refractory multiple myeloma (rrMM) who had received at least 2 prior myeloma regimens. Subjects received one cycle of CT053 CAR-BCMA after fludarabine/cyclophosphamide infusion. The primary objective was the assessment of subject safety. The secondary objectives were pharmacokinetics and efficacy. Efficacy was assessed according to IMWG 2016 criteria. The data cutoff date was June 30, 2019. Results: A total of 24 subjects with median age of 60.1 years (range, 38.5-69.9) were enrolled from Sep 10, 2017 to Sep 22, 2018 (Table 1). The subjects had a median of 4.5 (range, 2-11) prior regimens of therapy, and 41.7% (10/24) underwent autologous stem cell transplantation. At baseline, eleven out of 24 subjects (45.8%) had concomitant extramedullary involvement. Eight subjects (33.3%) had ECOG score 2-3, and 9 subjects (37.5%) reported ISS grade III. All subjects received lymphodepletion preconditioning of fludarabine/cyclophosphamide for 2-4 days, followed by one cycle of CT053 at a dose of 1.5 x 108 T cells except 3 subjects who received 0.5 x 108, 1 x 108, and 1.8 x 108 cells, respectively. The overall response rate was 87.5% (21/24) including 79.2% (19/24) with complete responses or stringent complete response (5 CR, 14 sCR). As shown in Figure 1, P1 who received the lowest dose of 0.5 x 108 experienced partial response (PR) at M1 and very good partial response (VGPR) at M2. P1 then converted to CR at M14 and sCR at M16. Among 20 subjects who underwent the evaluation of minimal residual disease (MRD) status, 17 achieved MRD-negative (≤10−4 nucleated cells) and reported a tumor response (17 CR/sCR). In 13 subjects with ongoing CR/sCR, the median follow-up after CT053 infusion was 383 days (range, 301-467). Nine subjects progressed with median progression-free period of 281 days (range, 57-573); among them, 5 progressed within 6-12 months, 1 at 13 months and 1 at 19 months. Compared to 13 subjects with persistent CR/sCR, the 9 progressed subjects were observed to have a higher percentage of ECOG score 2-3 (66.7% vs 15.4%), ISS Grade III (55.6% vs 15.4%), and concomitant extramedullary diseases (66.7% vs 38.5%) and a decreased hemoglobin (70g/L vs 92g/L) at baseline. Three subjects died of disease progression at the time of analysis. Hematologic toxic effects were the most common treatment-related adverse events of grade (G) 3 or higher, including white blood cell count decreased (87.5%), neutrophil count decreased (66.7%), lymphocyte count decreased (79.2%) and thrombocytopenia (25%). No dose limiting toxicities were observed. Low-grade cytokine release syndrome (CRS) was reported in 15 of 24 (62.5%) subjects. All CRS events (3 G1, 12 G2) recovered within 2-8 days; among them 8 received tocilizumab. Three subjects (12.5%) had neurotoxicity (2 G1, 1 reversible G3). One subject died of bone marrow failure and neutropenic infection. CAR-BCMA T cell expansion was detected as early as Day 1-7 after infusion and reached peak values on Day 7-21 with the highest at 4.5×105 copies/µg genomic DNA. Median T cell persistence was 172 days. The longest persistence of CAR-BCMA copies was measured at 341 days and continues. Conclusion: This study demonstrated that CT053 had an excellent efficacy and a good safety profile in subjects with rrMM. Disclosures Li: CARsgen Therapeutics Co. Ltd: Employment, Equity Ownership. Xiao:CARsgen Therapeutics Co. Ltd: Employment. WANG:CARsgen Therapeutics Co. Ltd: Employment. Yuan:CARsgen Therapeutics Co. Ltd: Employment. Ma:CARsgen Therapeutics Co. Ltd: Employment.

Description: The clinical value of plasma Epstein-Barr virus (EBV) DNA has not been evaluated in patients with early-stage extranodal nasal-type NK/T-cell lymphoma (NKTCL) receiving primary radiotherapy. Fifty-eight patients with stage I disease and 11 with stage II disease were recruited. High pretreatment EBV-DNA concentrations were associated with B-symptoms, elevated lactate dehydrogenase levels, and a high International Prognostic Index score. EBV-DNA levels significantly decreased after treatment. The 3-year overall survival (OS) rate was 82.6% for all patients. Stage I or II patients with a pretreatment EBV-DNA level of ≤ 500 copies/mL had 3-year OS and progression-free survival (PFS) rates of 97.1% and 79.0%, respectively, compared with 66.3% (P = .002) and 52.2% (P = .045) in patients with EBV-DNA levels of 〉 500 copies/mL. The 3-year OS and PFS rates for patients with undetectable EBV-DNA after treatment was significantly higher than patients with detectable EBV-DNA (OS, 92.0% vs 69.8%, P = .031; PFS, 77.5% vs 50.7%, P = .028). Similar results were observed in stage I patients. EBV-DNA levels correlate with tumor load and a poorer prognosis in early-stage NKTCL. The circulating EBV-DNA level could serve both as a valuable biomarker of tumor load for the accurate classification of early-stage NKTCL and as a prognostic factor.

Description: Background: Although type 1 von Willebrand disease (VWD) is the most common bleeding disorder seen by pediatric hematologists, making a definitive diagnosis continues to be a challenge in clinical practice. Both the International Society on Thrombosis and Haemostasis (ISTH) and the Hospital for Sick Children in Toronto (HSC) have proposed diagnostic criteria for type 1 VWD. These include abnormal laboratory values, significant mucocutaneous bleeding, and/or a positive family history. Most recently, the ISTH published updated recommendations, which differed only in the requirement of more abnormal laboratory results (VWF:Ag 5–20 IU/ml). We applied ISTH and HSC criteria, as well as updated ISTH criteria, to a large population of pediatric patients diagnosed with type 1 VWD. We hypothesized that a substantial number of patients would not meet either HSC or ISTH diagnostic criteria. Methods: We performed a retrospective medical record review of all type 1 VWD patients at our Hemostasis and Thrombosis Center. We evaluated each record for bleeding history, family history, and laboratory values. Frequencies of fit for HSC, ISTH and updated ISTH criteria were calculated. Mean VWF:Ag, VWF:RCo, and bleeding scores (Rodeghiero et al, J Thromb Haemost, 2006) were compared across populations meeting each proposed criteria. Results: Of 201 patients, 33.9% met the HSC definition of “definitive” type 1 VWD, 4.5% met ISTH definition, and 0% met updated ISTH definition. An additional 56.2% (HSC), 15.4% (ISTH), and 6% (updated ISTH) met definitions of “possible” type 1 VWD. For each proposed definition, criteria for significant mucocutaneous bleeding were most likely to be met, while criteria for abnormal laboratory values were least likely. In fact, 74% of patients had significant bleeding as defined by the HSC (56% as defined by ISTH). We did find significant clinical and laboratory differences between patients labeled as definite, possible, and normal by ISTH and HSC criteria. For example, patients meeting criteria for definite disease by HSC criteria had a mean bleeding score of 3.5 and mean VWF:Ag of 31 IU/ml, compared to 2.6 and 47 IU/ml in patients labeled as possible, and 2.2 and 68 IU/ml in patients labeled as normal (p=0.001 bleeding score,

Description: Loss-of-function mutations in Ten-Eleven-Translocation 2 (TET2) gene have been identified in various human myeloid and lymphoid malignancies. Recently, the TET gene family (TET1, TET2, and TET3) was found to function as DNA methylcytosine dioxygenase that is able to oxidize 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). In Tet2-deficient mouse models, Tet2 has been shown to play an important role in regulating self-renewal and differentiation of hematopoietic stem cells. These Tet2-deficient mice would gradually develop a chronic myeloid neoplasm resembling human chronic myelomonocytic leukemia suggesting that TET2 may function as a tumor suppressor. In the present study, we investigated the role of tet2 in zebrafish early hematopoiesis. During zebrafish early development, the expression of tet1, tet2, and tet3 by qRT-PCR can be detected mainly after the segmentation stage (26-somite), with fluctuated expression levels thereafter. Whole-mount in situ hybridization revealed that tet2 expression was strong over aorta-gonad-mesonephros region at 48 hours post-fertilization (hpf). Morpholino oligonucleotide (MO) knock-down of tet2 increased the expression of tet1, tet3, dnmt3aa, gata-1, alpha-Hb and fli1a (48 hpf) as well as rag2 and lck (4 days post-fertilization), and the expression of spi1b and mpo decreased (48 hpf). The expression of primitive hematopoietic stem cell markers scl and lmo2, as well as dnmt3ab, beta-Hb, l-plastin, and rag1 were unaffected. The levels of 5-mC and 5-hmC measured by ELISA were also decreased after MO knock-down of tet2. The number of gata-1 expressing red blood cells increased after tet2 MO knock-down as evaluated by flow-cytometry indicating that tet2 deficiency increased erythropoiesis. These preliminary results suggest that tet2 might play a role in the epigenetic regulation of zebrafish early hematopoiesis including erythropoiesis. Recently, transcription activator-like effector nuclease (TALEN) has been shown to generate targeted genomic editing in zebrafish. To validate our observation, we therefore utilized customized TALENs pair to generate tet2 knock-out zebrafish animal model. We designed a pair of TALENs targeting first exon of tet2 and our tet2 TALENs were able to generate insertion and/or deletion in targeted region of tet2 exon 1 in 25% to 44% zebrafish embryos. We obtained a total of fifteen different tet2 mutation genotypes F1 fish, and seven of them were predicted to cause early termination of transcription. The in-cross of these F1 genotypes matched the Mendelian inheritance. The tet2-/- knock-out F2 zebrafish is not embryonic lethal and can grow to sexually mature adult fish. The detailed analysis of tet2-/- knock-out zebrafish early hematopoiesis will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3760 Lentiviral vectors (LV) assure stable transgene expression in vivo, allowing to investigate genes and their functions. In recent years, lentiviral gene transfer was considered to facilitate the generation of transgenic mice with a higher yield of transgenic offspring as compared to commonly used DNA microinjection. We applied LV to generate a mouse model transgenic for SETBP1 and eGFP. Murine zygotes were infected at dE0.5 with lentiviral particles directly injected into the perivitelline space. Specific PCRs for either the SETBP1 transgene or for the WPRE element of the lentiviral construct verified complete lentiviral integration in newborn pups (F0). Lentiviral integration sites were detected using highly sensitive LAM-PCR in 65% of 31 analyzed F0 mice. Germline transmission was shown in a total of 33% vector positive offspring from 5 out of 9 F0 mice. However, no ectopic transcription and overexpression of neither SETBP1 nor eGFP could be detected in transgenic mice. We therefore analyzed the methylation status of the internal SFFV promoter (SFFVp) by bisulfite sequencing. Extensive methylation (around 90%) could be assessed in 18 of 18 analyzed CpGs within the promoter region in F0 animals and in all progeny determined (n=12). We transduced mES cells with LV.SFFV.Setbp1.IRES.eGFP or the corresponding eGFP-expressing control vector to exclude transgene effects on epigenetic silencing of SFFVp sequences in self-inactivating LVs. Differentiation of ES cells infected with the transgene vector and SFFV driven control vector led to a 1.8 – 3.5 fold decrease of eGFP expression. To analyze whether methylation of SFFVp sequences is a common event even in adult tissues, we analyzed the methylation status of peripheral blood in mice transplanted with bone marrow cells transduced with either gammaretroviral vectors (RV) or LV 3 months after transplantation (n=7). Interestingly, SFFVp sequences in peripheral blood of mice transplanted with LV transduced bone marrow were stronger methylated than CpGs of SFFVp in RV transplants. Our data demonstrate that the commonly used SFFV promotor is highly methylated with remarkable strength and frequency during development in vivo and differentiation in vitro. We conclude that lentiviral vectors using an internal SFFV promoter are not suitable for the generation of transgenic mice or constitutive expression studies in hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

Description: The transcription factor NF-E2 is overexpressed in the vast majority of patients with Myeloproliferative Neoplasms (MPN). In Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) NF-E2 levels are elevated independent of the presence or absence of the JAK2V617Fmutation. We have recently shown that NF-E2 overexpression in a murine model leads to an MPN phenotype followed by spontaneous transformation to acute leukemia in a subset of mice. We have demonstrated that increased NF-E2 transcription is mediated by the transcription factor and proto-oncogene AML-1, which is overexpressed in MPN patients, again irrespective of the JAK2V617Fstatus. AML-1 binds to a conserved enhancer region located 3500 bp upstream of the NF-E2 transcriptional start site. Since AML-1 responsive genes are often also inversely regulated by the tumor suppressor AML-2 (RUNX3), we investigated whether NF-E2 expression is affected by AML-2 as well. Using chomatin immunoprecipitation assays (ChIP) we show that AML-2 binds the NF-E2 enhancer in vivo at a site distinct from but in proximity to the three AML-1 sites in the -3500bp region. AML-2 binding strongly represses transcription off the NF-E2 enhancer in reporter gene assays. This repression is abrogated by site directed mutagenesis of the AML-2 recognition sequence. Likewise, lentivirally induced AML-2 expression drastically reduces the amount of NF-E2 protein in erythroid and myeloid cells. These data clearly demonstrate that NF-E2 expression is directly regulated by AML-2. AML-2 thus serves as a repressor on the hematopoietic NF-E2 gene, a function previously noted mainly in solid tumors. In primary cells from patients with polycythemia vera (PV) AML-2 mRNA expression is significantly reduced. Moreover, ChIP assays demonstrate that in primary PV cells, significantly less AML-2 is bound to the NF-E2 enhancer than in healthy controls. Decreased repression by AML-2 thus cooperates with increased AML-1 induced transcription to elevate NF-E2 levels in PV patients. It has been demonstrated that AML-2 expression can be regulated by two distinct epigenetic mechanisms. For one, DNA methylation of the AML-2 promoter has been reported to silence AML-2 expression in gastric, colorectal and bladder cancers. On the other hand, aberrant histone methylation in the promoter region can also silence AML-2 expression. Here we show that DNA methylation of the AML-2 promoter is unaltered in PV patients. Rather, PV patients display aberrant histone methylation in the AML-2 promoter. Compared to healthy controls, H3K27me3 is significantly increased and H3K4me3 is significantly decreased in primary PV cells. This results in an inactive chromatin conformation on the AML-2 promoter in PV patients. Moreover, we show here that the histone-lysine-methyl-transferase “enhancer of zeste homologue 2” (EZH2), which confers the K3K27me3 mark, binds to the AML-2 promoter and decreases AML-2 expression. PV patients demonstrate significantly increased levels of EZH2 binding to the AML-2 promoter Treatment of primary PV cells and MPN cell lines with 2'-deoxy-5-azacytidine (DAC, Decitabine) ex vivo reverses the altered histone methylation, restoring the pattern found in healthy controls and decreases EZH2 binding to the AML-2 promoter. At the same time, Decitabine treatment induces AML-2 protein expression, decreases EZH2 expression and reduces the elevated NF-E2 levels. Moreover, a post-PV AML patient receiving Decitabine, displayed normalization of AML-2 promoter histone methylation and EZH2 binding on day 8 and day 15 of treatment. Taken together our data demonstrate that epigenetic silencing of AML-2 contributes to the elevated NF-E2 expression observed in MPN patients. Treatment with Decitabine restores physiological histone modifications to the AML-2 locus and decreases NF-E2 levels by reactivating AML-2 expression. These data provide a molecular rationale for extending the clinical investigation of epigenetic modifiers such as Decitabine or Azacitidine, currently used in MDS and AML, to patients with MPNs. A phase I study using Azacitidine in high risk PMF patients is currently being planned by the MPD-RC. Disclosures: No relevant conflicts of interest to declare.

Description: Background: The efficacy and safety of rituximab(R)-based immunochemotherapy, the standard regimens for patients (pts) with diffuse large B-cell lymphoma (DLBCL) which is more common in Asia than in Western countries, are well confirmed in RCT studies. However, the safety and effectiveness of R+chemo for DLBCL in real world use is not widely reported, especially population are normally excluded in RCT studies. The objective of this observational study is to investigate the safety and effectiveness of R+chemo as 1st line treatment for Chinese DLBCL in routine clinical practice. Methods: This study was a multicenter, prospective, single-arm observational study conducted in China. DLBCL pts eligible to receive R+chemo (CHOP or non-CHOP) as 1st line treatment were enrolled with no specific exclusion criteria. The primary endpoint was safety. Data on safety and effectiveness were collected from medical records 120 d after the last R dose was administered. This study was registered in clincialtrials.gov (NCT01340443). Results: In total, 279 pts (162male/117femal) with a median age of 57 yrs (range 13 to 88 yrs) were included in the safety analysis set. By IPI criteria, 50.2% of pts were low risk, 25.4% low-intermediate risk, 16.5% intermediate-high risk, 7.2% high risk and 0.7% with unknown risk. The most common concomitant diseases observed are liver (44,15.8%), heart (23, 8.2%), kidney (9, 3.2%) or lung (7, 2.5%) disease. Pts received R+chemo treatment with a median cycle of 6 and a median interval of 24 d. In total, 52.7% pts had grade 3-4 AEs and 16.8% pts had SAE (Table 1). AE related death was 1.1% (n=3). 67.0% pts had any grade hematologic toxicity and the most common grade 3/4 hematologic toxicities were leukocytopenia (29.7%), erythrocytopenia (3.9%), and thrombocytopenia (5.7%). Infection (46.2%), gastrointestinal toxicity (45.2%), and liver toxicity (12.5%) were common non-hematologic toxicities. The AEs of special population (with common concomitant and very young/older DLBCL) are listed in Table 2. For HBV management, the incidence of HBV reactivation in HBsAg+, HBsAg-/HBcAb+, HBsAg-/HBcAb-, and undefined pts was 12.5% (3/24), 4.3% (3/69), 0.7% (1/149), and 2.7% (1/37), respectively, no death due to HBV reactivation 120 d after the last R dose was administered. The detail outcomes of HBV reactivation management in this study was reported in EHA 2014. For ITT population (n=258), the overall response rate was 93.7%. Rates of complete response (CR), unconfirmed CR (CRu) and partial response (PR) were 55.0%, 18.2% and 20.9%, respectively. Summary and Conclusions: During this observation study, the incidence of adverse events of R+chemo as 1st line for DLBCL in real world were tolerable and consistent to previous reports. The AEs in special DLBCL sup-population (very younger, older or with common concomitant disease) are well tolerated too. R+chemo treatment brought more than 90% response rate in these Chinese pts might due to the pts with relative low IPI score. More education on standard management of HBV is needed. Accordingly, this real world study further validates the safety and effectiveness of using R+chemo to treat pts with DLBCL. Table 1: The Hamatologist and common non-hematologist AEs ¡¡ Any grade, n (%) Grade 3/4, n (%) SAE, n (%) Death, n (%) Any toxicity 267 (95.7) 147 (52.7) 47 (16.8) 3 (1.1) Hematological toxicity 187 (67.0) 104 (37.3) 6 (2.2) 0 (0.0) Bone Marrow Failure 23 (12.3) 14 (7.5) 2 (1.1) 0 (0.0) Leukocytopenia 167 (59.9) 83 (29.7) 4 (1.4) 0 (0.0) Erythrocytopenia 62 (22.2) 11 (3.9) 0 (0.0) 0 (0.0) Thrombocytopenia 24 (8.6) 16 (5.7) 1 (0.4) 0 (0.0) Common Non-hematological toxicity Infection 129 (46.2) 48 (17.2) 34 (12.2) 2 (0.7) Gastrointestinal toxicity 126 (45.2) 7 (2.5) 2 (0.7) 0 (0.0) Liver toxicity(SMQ) 663(22.6) 10 (3.6) 2 () 0 (0.07) Cardiac toxicity(SMQ) 29 (10.4) 4 (1.4) 3 (1.1) 1 (0.4) Kidney toxicity 9 (3.2) 1 (0.4) 0 (0.0) 0 (0.0) Table 2: The AEs of special population (with common concomitant and very young/older DLBCL) ¡¡ Total common concomitant disease ¡¡ Age ¡¡ Cardiac History Liver History ¡¡ =80 19-79 ¡¡ (N=279) (N=23) (N=44) ¡¡ (N=10) (N=269) ¡¡ No.% No.% No.% ¡¡ No.% No.% AE 267 (95.7) 22 (95.7) 42 (95.5) ¡¡ 10 (100) 257 (95.5) SAE 47 (16.8) 7 (30.4) 11 (25.0) ¡¡ 3 (30.0) 44 (16.4) AESI 46 (16.5) 6 (26.1) 9 (20.5) ¡¡ 2 (20.0) 44 (16.4) ADR 226 (81.0) 21 (91.3) 36 (81.8) ¡¡ 10 (100) 216 (80.3) Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1065 Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that controls cell proliferation, differentiation, apoptosis, immune responses, and tissue fibrosis. Circulating platelets contain a high concentration of TGF-β1 in their α -granules and release it as an inactive (latent) complex when activated. We previously reported that platelet TGF-β1 is responsible for ∼45% of basal plasma TGF-β1; that shear force can activate TGF-β1 in vitro; and that mice with megakaryocyte-specific deletion of TGF-β1 [PF4-Cre/Tgfb1flox (Tgfb1flox)], which have ∼15% of normal platelet TGF-β1, are partially protected from developing cardiac hypertrophy, fibrosis, and systolic dysfunction induced by transverse aortic constriction (TAC) surgery. Since the TAC model involves placing a constriction distal to the innominate artery, which produces high shear both at the site of stenosis and in innominate artery, in this study we tried to simulate better the hemodynamics of human valvular aortic stenosis by constricting the ascending aorta (AAC) of both C57Bl/6 wild-type (WT) mice and Tgfb1floxmice. The AAC surgery employed a 27 gauge needle guide and reduced the diameter of the ascending aorta by 〉80% in both WT and TGFb1flox mice (p