ALBERT

Description: Key Points The coagulation protease FVIIa attenuates TNF-α- and LPS-induced inflammation both in vitro and in vivo via an EPCR-dependent mechanism. FVIIa-EPCR-PAR1-mediated anti-inflammatory signaling transmits through the β-arrestin-1-dependent pathway.

Description: Introduction Effective therapies for R/R AML remain limited. MEK or MDM2 inhibition can downregulate MCL1, overcoming resistance to BCL2 inhibition. Preclinical synergy was seen when combining BCL2 inhibitor Ven with MEK inhibitor cobimetinib (cobi) or MDM2 inhibitor idasa (Han et al. ASH 2016; Pan et al. Cancer Cell 2017), supporting clinical evaluation in AML. Preliminary data in a Phase Ib dose-escalation study (NCT02670044) evaluating Ven+cobi/idasa in R/R AML suggested both combinations were tolerable (Daver et al. ASH 2017). However, Ven+cobi was closed due to limited clinical activity. Here we present data for additional pts, longer follow-up and biomarker analyses for Ven+idasa. Methods This ongoing, open-label, multicenter study evaluates safety, tolerability and efficacy of Ven+idasa in R/R AML or secondary AML previously treated for an antecedent hematologic disease. Pts 〉60 yrs of age and ineligible for cytotoxic therapy/allogeneic stem cell transplant were enrolled. A 2-dimensional dose escalation was used to establish the maximum tolerated dose: pts received doses of Ven orally (PO) daily (400mg or 600mg) + idasa PO daily on Days 1-5 (150mg, 200mg, or 400mg) in 28-day cycles. Plasma samples were taken for PK analysis at Cycles 1 and 2 Days 1 and 5, and Cycle 4 Day 1. BCL2, BCLxL and MCL1 status and minimal residual disease (MRD) were assayed centrally at Covance Laboratories using multicolor flow cytometry. Mutation (mut) sequencing was performed by Foundation Medicine using FoundationOne Heme at screening and from last bone marrow collected on study. Results As of April 6 2018, 34 pts received Ven+idasa across all dose cohorts (Table 1). Median age: 74 (range 64-93) yrs; median prior therapies: 1 (range 1-4); ECOG performance status 2: 18%; refractory: 56%; secondary AML: 53%; adverse cytogenetics: 27%. Pre-therapy mut data were available for 32 pts; most common muts were RUNX1 14 (41%), ASXL1 11 (32%), SRSF2 11 (32%). Other significant pre-therapy muts: TP53 6 (18%), IDH2 7 (21%), IDH1 1 (3%), FLT3 4 (13%). The most common adverse events (AEs) were diarrhea (88%) and nausea (71%); the most common grade (Gr) ≥3 AEs were neutropenia (32%), febrile neutropenia (32%), thrombocytopenia (29%; Table 2). After 2 cases of Gr 3 diarrhea in the Ven 600mg cohorts, mandatory prophylaxis was implemented; no further cases of Gr ≥3 diarrhea were seen in the following 10 pts. Laboratory tumor lysis syndrome occurred in 3 pts (9%); none required treatment discontinuation. There was no apparent PK drug-drug interaction between Ven and idasa. PK was dose-proportional over the ranges tested for Ven and idasa. The recommended Phase II dose (RP2D) has not been identified yet. Across all dose cohorts, 30/34 pts were response-evaluable; the remaining 4 were still on study treatment without post-baseline response assessment. The anti-leukemic response rate (CR+CRp+CRi+MLFS+PR) was 37% (11/30). Across the 2 Ven 600mg cohorts, which are being considered for RP2D, the anti-leukemic response rate was 9/18 (50%) (Table 1, Figure 1). MRD negativity (

Description: Tissue factor (TF), an integral membrane glycoprotein, is a cofactor for coagulation factor VIIa (FVIIa) and primary cellular initiator of the coagulation. Upon vascular injury or in disease conditions, blood comes in contact with TF, and the formation of TF-FVIIa complex initiates activation of the coagulation cascade. While TF is critical for the maintenance of hemostasis, aberrant expression of TF activity could lead to thrombotic disorders. Typically, most of TF on cell surfaces exist in a cryptic, coagulant inactive state, and an "activation" step (decryption) is essential for the transformation of cryptic TF to prothrombotic TF. Our recent studies showed that sphingomyelin (SM) in the outer leaflet of the plasma membrane is responsible for maintaining TF in an encrypted state in resting cells. The hydrolysis of SM, by either bacterial sphingomyelinase (bSMase) or acid-sphingomyelinase (ASMase) translocated from lysosomes to the outer leaflet in response to ATP, LPS or cytokine stimulation, increased TF activity on intact cells without altering TF protein levels. SM hydrolysis also led to the release of TF+ microvesicles (MVs). Inhibition of ASMase by functional inhibitors blocked LPS-induced TF procoagulant activity without impairing LPS-induced TF antigen levels in both in vitro and in vivo model systems. SM levels in the plasma membrane are regulated primarily by SM synthesizing enzymes, such as sphingomyelin synthases (SMS) 1 and 2 or SM hydrolyzing enzymes, such as ASMase and neutral SMases (nSMase). Many disease conditions, including diabetes, ischemia/hypoxia, and cancer, alter SM metabolism by altering the activities of the above enzymes. These diseases are also known to have increased thrombotic risk. To investigate the importance of SM metabolism in regulating TF procoagulant activity through TF encryption and decryption, we either overexpressed or silenced the enzymes involved in SM metabolism and determined their effect on TF procoagulant activity on intact cells and the release of TF+ MVs. Human monocyte-derived macrophages (MDMs) or human embilical vein endothelial cells (HUVEC) were chosen as cell model systems. In the first set of experiments, MDMs were transfected with adenovirus encoding SMS1, SMS2, or both to overexpress SMS. Analysis of SM levels in the outer leaflet by confocal microscopy and flow cytometry using SM specific binding protein (lysenin) revealed that overexpression of SMS1 or SMS2 increased SM levels in the outer leaflet. Measurement of TF activity on intact cells showed that overexpression of either SMS1 or SMS2 reduced both basal TF activity and the extent of increased TF activity following ATP or bSMase treatment. Overexpression of SMS1 or SMS2 also decreased the release of TF+ MVs. Overexpression of SMS1 or SMS2 had no significant effect on TF antigen levels. In the next set of experiments, MDMS were transfected with control scrambled RNA (scRNA) or siRNA specific for ASMase, nSMase1, nSMase2, or nSMase3. As expected from our earlier studies, ASMase silencing attenuated both basal and ATP-induced increased TF activity in MDMs. In case of nSMases, the knock-down of nSMase2 or nSMase3, but not nSMase1, reduced basal TF activity as well as ATP-induced TF decryption in MDMs. Analysis of SM levels in the outer leaflet showed that silencing of ASMase, nSMase2, or nSMase3 enhanced the SM content. The knock-down of either ASMase or nSMases did not affect TF antigen levels. In additional studies, HUVECs were transfected with control scRNA or siRNA specific for nSMase1, nSMAse2, or nSMase3. Forty eight hour post-transfection, HUVECs were stimulated with TNFα (10 ng/ml) plus IL-1β (10 ng/ml) for 6 h to induce TF expression. Analysis of cell surface TF activity showed that silencing nSMase2 or nSMase3, but not nSMase1, attenuated TNFα+IL-1β-induced TF procoagulant activity without decreasing TNFα+IL-1β-induced TF antigen levels. Overall, our data support the hypothesis that alterations in SM metabolism regulate TF procoagulant activity through encryption and decryption. Disclosures Rao: Takeda: Research Funding.

Description: Introduction Although CLL minimal residual disease (MRD) status is used in contemporary clinical trials aimed at maximizing response or determining treatment duration, its role as a predictive factor for PFS has only been established following chemoimmunotherapy. In contrast, whether MRD is a valuable tool for treatment choice in the era of novel targeted agents for CLL is unknown. Unlike kinase inhibitors, the BCL2 inhibitor venetoclax does result in undetectable MRD (uMRD). MURANO demonstrated significant PFS benefit for venetoclax + rituximab (VenR) given for a fixed duration vs bendamustine + rituximab (BR) in relapsed/refractory (R/R) CLL. Here we present analysis of peripheral blood (PB) MRD kinetics in relation to PFS in MURANO with long follow up, when all pts have completed therapy. Methods Pts were randomized to VenR (Ven 400mg/d for 2 yrs + R for first 6 mo) or BR (6 mo). Response was assessed clinically using complete blood count and physical exam at follow-up visits. PB MRD was analysed centrally by ASO-PCR and/or flow cytometry at Cycle 4, end of combination therapy (EOCT; mo 9) and every 3 mo thereafter until 3 yrs, then every 6 mo. As strong concordance between PB and bone marrow (BM) MRD with VenR was previously shown (Hillmen et al. ASCO 2018), we focus on PB MRD. Pts were categorized into uMRD (

Description: Introduction The measurement, analysis, and communication of treatment-related symptoms and their impact on the functioning of individuals in clinical trials is an area of interest to the medical community, the Food and Drug Administration and other regulatory agencies. Inclusion of GAs and PROs in early phase trials can characterize the heterogeneity of aging and guide development of therapies to benefit older adults. Despite evidence that GAs and PROs are feasible and predictive of outcome, few early phase trials have integrated them to inform on treatment tolerance. The aim of this analysis is to describe changes in GAs and PROs among older adults with R/R AML in a Phase 1b trial of BCL2 inhibitor venetoclax (Ven) with either MEK inhibitor cobimetinib (cobi) or MDM2 antagonist idasanutlin (idasa) (NCT02670044). Methods Patients (pts) aged ≥60 yrs who were ineligible for cytotoxic therapy/allogeneic stem cell transplant were enrolled in this open-label, multicenter study, designed to evaluate the safety, tolerability, and efficacy of Ven+cobi/idasa in R/R or secondary AML. GA measures were Short Physical Performance Battery (SPPB; gait speed, balance tests, chair stands), Hematopoietic Cell Transplant-Comorbidity Index (HCT-CI) and Short Blessed Orientation Test (SBOT). Treatment-related symptoms were assessed via the PRO version of the Common Terminology Criteria for Adverse Events (PRO-CTCAE), administered electronically throughout treatment. GAs were administered at screening, Cycle (C) 2 Day (D) 1, C3D1, and C7D1; PRO-CTCAE was administered at C1D1, weekly during C1, for the first 3 weeks of C2 and C3, and monthly starting at C4D1. Mean GA scores were calculated over time for each treatment arm and pt-level heatmaps were created. The 'frequency', 'severity', 'interference', and 'presence' of PRO-CTCAE symptoms were reported at each assessment, with a longitudinal analysis to understand how symptoms changed over the course of treatment. Results As of April 6 2018, 64 pts had been treated with either Ven+cobi or Ven+idasa. Baseline characteristics were comparable between arms; respectively: median age, 72, 74 yrs; ECOG performance status 0-1, 83%, 82%; refractory disease, 63%, 56%. GA and PRO data were available in 52 and 38 pts, respectively. Screening of GAs identified a physically frail (mean SPPB score 75% and 〉50% reported 'none' or 'mild' decreased appetite and fatigue, respectively. In both arms, pt numbers reduced over C1-3, with 8 pts in the Ven+cobi and 6 pts in the Ven+idasa arms providing data at C3D1; this was largely due to attrition on study. Pts reported that their worst post-baseline symptom was 'frequent' or 'almost constant' symptoms of nausea (55%), diarrhea (71%), and vomiting (5%) at some point, and 'severe' or 'very severe' decreased appetite (55%) or fatigue (55%). Variations in the worst post-baseline symptoms were observed between treatment arms, although this was not statistically tested. Symptoms tended to increase and decrease with the start and end of each treatment cycle and appeared worse during C1 for Ven+cobi (Figure 1). Despite differences in symptoms during the cycle, there did not appear to be changes in mean SPPB scores from baseline to C2, no cognitive impairment during treatment, and little to no increase in comorbidity burden. These results may be influenced by a reduction in pt numbers in each treatment arm over subsequent assessments. Conclusion PROs from this Phase 1b study indicate an increased frequency of treatment-related symptoms, in particular, nausea, diarrhea, vomiting, decreased appetite and fatigue, in older AML pts treated with Ven+cobi or Ven+idasa compared with pre-treatment. PROs and GAs can provide additional complementary insight into the older pt's treatment experience on early phase clinical trials that can inform design of later-phase studies. However, strategies to address missing data will be important for maximizing value gained. Disclosures Klepin: Genentech Inc: Consultancy. Campinha-Bacote:Genentech Inc: Employment. Hong:Genentech Inc/Roche: Employment, Other: Ownership interests PLC. Phuong:Genentech Inc: Employment, Equity Ownership, Other: Ownership interests PLC. Wang:Genentech Inc: Employment; F. Hoffmann-La Roche Ltd: Equity Ownership. Mobasher:F. Hoffmann-La Roche Ltd: Other: Ownership interests non-PLC; Genentech Inc: Employment. Trask:Genentech Inc: Employment; F. Hoffmann-La Roche: Employment.

Description: A novel glass needle–mediated microinjection method for delivery of macromolecules, including proteins and larger transgene DNAs, into the nuclei of blood stem/progenitor cells was developed. Temporary immobilization of cells to extracellular matrix–coated dishes has enabled rapid and consistent injection of macromolecules into nuclei of CD34+, CD34+/CD38−, and CD34+/CD38−/Thy-1lo human cord blood cells. Immobilization and detachment protocols were identified, which had no adverse effect on cell survival, progenitor cell function (colony forming ability), or stem cell function (NOD/SCID reconstituting ability). Delivery of fluorescent dextrans to stem/progenitor cells was achieved with 52% ± 8.4% of CD34+ cells and 42% ± 14% of CD34+/CD38−cells still fluorescent 48 hours after injection. Single-cell transfer and culture of injected cells has demonstrated long-term survival and proliferation of CD34+ and CD34+/CD38−cells, and retention of the ability of CD34+/CD38− cells to generate progenitor cells. Delivery of DNA constructs (currently ≤ 19.6 kb) and fluorescently labeled proteins into CD34+ and CD34+/CD38− cells was achieved with transient expression of green fluorescent protein observed in up to 75% of injected cells. These data indicate that glass needle–mediated delivery of macromolecules into primitive hematopoietic cells is a valuable method for studies of stem cell biology and a promising method for human blood stem cell gene therapy.

Description: Introduction Venetoclax (Ven) is a highly selective oral inhibitor of BCL2, a key regulator of the intrinsic apoptotic pathway, which is constitutively overexpressed in CLL. Efficacy and safety of VenR given for a fixed duration in R/R CLL was evaluated in the randomized Phase III MURANO study compared with standard bendamustine + rituximab (BR). A first pre-planned analysis, when the majority of patients (pts) were still on study treatment (median follow-up 23.8 mo), established a superior PFS of VenR over BR (Seymour et al. NEJM 2018). With all pts having completed therapy, we analysed long-term outcome with a median follow up of 36.0 mo. Methods Pts were randomized to receive 6 cycles of VenR followed by Ven 400mg once daily for a total of 2 yrs, or 6 cycles of BR. Disease status was assessed by CT scan at screening, Cycle (C) 4 and 2−3 mo after end of combination therapy (EOCT). During Ven single agent and at follow-up visits, response was determined by clinical assessment every 3 mo until 3 yrs of follow-up, then every 6 mo thereafter or until PD. PFS status was based on investigator assessment. Peripheral blood (PB) minimal residual disease (MRD) was analysed centrally by ASO-PCR and/or flow cytometry at C4, EOCT and every 3/6 mo thereafter. All p values are descriptive. Results In total, 389 pts were enrolled in VenR (n=194) or BR (n=195) arms. As of May 8 2018, all pts were off treatment. For BR, 154 (79%) had completed 6 cycles. In the VenR arm, 174 (90%) completed the VenR combination phase and 130 (67%) had completed 2 yrs of Ven. The remainder had PD (11%), died (1%), or withdrew due to AEs (15%) or other reason (6%). Median Ven exposure duration and relative dose intensity were 24.4 (range 0-27.9) mo and 97.4% (26-100%), respectively, overall and 17.7 (0.5-21.9) mo and 99% during Ven single-agent therapy. With a median time off Ven after 2 yrs treatment of 9.9 (1.4-22.5) mo, PFS with VenR remains superior to BR (HR, 0.16 [95% CI 0.12, 0.23]; p1% in PB at Mo 24 (when Ven single agent was scheduled to cease) and 10/16 pts had del(17p)/TP53 mutation at baseline. Clinical and cytogenetic risk factors in pts with and without PD after Mo 24 are in Table 1. In this analysis, OS improvement was seen with VenR over BR (HR, 0.50 [95% CI 0.30, 0.85]; p=0.0093; 3-yr rate: 87.9% vs 79.5%; Figure 4). Subsequent CLL-directed treatment was given after PD in 91 pts in the BR arm. 71/91 (78%) BR arm pts received novel targeted agents, including 45 who had ibrutinib and 7 who had Ven. 27/194 (14%) pts in the VenR arm received subsequent therapy: 13/27 (44%) had novel targeted agents as next treatment, including 8 pts who had ibrutinib and 3 who were re-treated with Ven. See Table 2 for a safety overview for VenR combination and Ven single-agent periods. During Ven single agent: 17/171 pts (10%) had an AE leading to drug withdrawal; 7/171 pts (4%) had an AE leading to dose reduction; 44/171 pts (26%) had a Ven dose interruption due to an AE; 7/171 pts (4%) had a fatal AE (4 other cancers, 2 cardiac, 1 pneumonia). Grade 3-4 AEs occurred in 59/171 pts (35%); the most frequent were neutropenia (20 pts, 12%), anemia (5 pts, 3%), and thrombocytopenia (3 pts, 2%). 12/171 (7%) pts had a grade 3-4 infection in the Ven single-agent phase. The total number of Richter transformation events was 7 with VenR and 6 with BR. Conclusions With all pts off treatment and 3 yrs' median follow-up, continued substantial benefit was observed with VenR, with PFS and OS superior to BR. There were no new safety signals; most pts were able to complete treatment. The rate of CLL progression in the first 12 mo after Ven completion was modest (13%), supporting the feasibility and safety of a time-limited VenR duration. The protocol has been amended to include assessment of response and durability of disease control with VenR reintroduction at PD. This updated analysis of this Phase III global randomized study demonstrates clinically meaningful benefit of the VenR chemotherapy-free regimen with a fixed duration in all pts with R/R CLL. Disclosures Seymour: AbbVie: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Consultancy. Kipps:Genentech Inc: Consultancy, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Celgene: Consultancy; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Eichhorst:AbbVie, Celgene, Gilead, Janssen, Mundipharma, Novartis, Roche: Honoraria, Other: Travel support, Research Funding. Hillmen:Alexion Pharmaceuticals, Inc: Consultancy, Honoraria; Gilead Sciences, Inc.: Honoraria, Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Novartis: Research Funding; Celgene: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Assouline:Pfizer: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; BMS: Honoraria, Research Funding, Speakers Bureau. Owen:Teva: Honoraria; Celgene: Research Funding; F. Hoffmann-La Roche Ltd: Honoraria, Research Funding; Merck: Honoraria; AbbVie: Research Funding; Janssen: Honoraria, Research Funding; Pharmacyclics: Research Funding; AstraZeneca: Honoraria, Research Funding. Robak:Janssen: Consultancy, Honoraria; Gilead: Consultancy; AbbVie, Inc: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria. de la Serna:Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Jaeger:Takeda-Millenium: Membership on an entity's Board of Directors or advisory committees; Takeda-Millenium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; MSD: Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Infinity: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Cartron:Gilead Sciences: Honoraria; Celgene: Consultancy, Honoraria; Janssen: Honoraria; Roche: Consultancy, Honoraria; Sanofi: Honoraria. Montillo:Gilead: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding. Lamanna:Acerta: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jannsen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Verastem: Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Verdugo:AbbVie, Inc: Employment, Equity Ownership. Punnoose:Genentech Inc: Employment; Roche: Equity Ownership. Jiang:Genentech Inc: Employment, Equity Ownership. Wang:Genentech Inc: Employment; F. Hoffmann-La Roche Ltd: Equity Ownership. Boyer:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Humphrey:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Mobasher:F. Hoffmann-La Roche Ltd: Other: Ownership interests non-PLC; Genentech Inc: Employment. Kater:Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Key Points tmTNF-α expressed on LSC and leukemia cells correlates with poor risk stratification and adverse clinical parameters. Targeting tmTNF-α by monoclonal antibody eradicates LSC and blasts, preventing leukemia regeneration in secondary transplant in NOD-SCID mice.

Description: Background : FLT3-ITD mutation remains a great challenge in treating acute myeloid leukemia (AML) patients, for the high incidence of early relapse and drug-resistant mutations to tyrosine kinase inhibitors (TKIs). The FLT3-ITD mutation results in a significant and sustained activation of downstream signaling pathways. However, the physiological negative regulators of those aberrantly activated pathways have not been fully elucidated. In this study, we aim to identify and exploit a key molecule directly interacting with FLT3 receptor and down-regulating FLT3 associated pathways. Methods: The potential proteins interacting with FLT3 were immunoprecipitated (IP) and screened by mass spectrometry. The binding of phosphatase SHP-1 and FLT3 was verified using co-IP and in situ proximity ligation assay (PLA) in cell lines and primary samples. The phosphatase activity and phosphorylation level of SHP-1 was determined by pNPP phosphatase activity assay and western blot. Biological effects of overexpressed SHP-1 by lentiviral vectors in AML cell-lines were assessed using proliferation, apoptosis and clonal formation assays in vitro and xenografts models in vivo. Plasmids of truncated or mutated structure of SHP-1 were constructed and overexpressed in cells to identify the key domain responsible for binding with FLT3 receptor. An artificial trans-membrane peptide was designed to activate SHP-1 activity and promote apoptosis in AML cells. Results: The researchers performed immunoprecipitations (IP) followed by LC-MS/MS based screen in human leukemia cell line MV4-11 for three times, and found that the phosphatase SHP-1 binds to FLT3. In addition, the interaction between SHP-1 and FLT3-WT or FLT3-ITD was verified by co-IP and in situ proximity ligation assay (PLA) in AML cell lines, primary samples and overexpression model of 293-FT.Partial results are showed in Figure A. Furthermore, in heterozygous cell line molm13 and engineered FLT3-ITD knock-in cell line SKM-1 by crispr/cas9 techonology, combined with PLA, pNPP phosphatase activity assay and western blot, we found that SHP-1 may bind with p-FLT3 and acquired phosphatase activity(Figure B-D). In turn, p-FLT3 itself and some important proteins in the downstream pathway would be dephosphorylated and the signal transmission would be terminated. In FLT3-ITD cells, the phosphorylation level of SHP-1 significantly reduced. By using lentivirus to overexpress or knockdown shp-1 protein in FLT3-WT and FLT3-ITD cells, the biological effects were studied. Experiments on cell apoptosis, proliferation and clonal formation have shown that overexpression of SHP-1 in AML cells may promote apoptosis, inhibit proliferation and clone formation, especially in FLT3-ITD cells. On the contrary, inhibition of shp-1 could promote proliferation and increase the ability of clone formation. Furthermore, DOX-inducible overexpression cell line of SHP-1 was obtained by tet-on virus system and the western blots showed that SHP-1 may inhibit p-ERK and p-STAT5 activity. In vivo experiments of NCG mice showed that, the degree of leukemia infiltration in peripheral blood was decreased in AML transplantation mice induced by DOX to express SHP-1 when compared to control group. Next, according to the analysis of protein structure by computer simulation, we constructed truncated and mutated plasmids and overexpressed in cells, then using PLA to verify the binding , we found that FLT3 interact with N-SH2 domain of SHP-1,exactly the R30 site(Figure E). Therefore, by using specific trans-membrane peptide, the researchers found that it could activate SHP-1 and promote apoptosis in AML cells, especially FLT3-ITD ones, inhibit cell proliferation and the downstream signal pathway of FLT3.Lastly,injection of peptide in subcutaneous tumor model of nude mouse showed that the tumor was significantly smaller than that of the control group(Figure F). Conclusion: We identify a negative regulator, namely phosphatase SHP-1, which directly interacts with FLT3 receptor through the N-SH2 domain and down-regulates FLT3 downstream signaling pathways. Overexpression of SHP-1 promotes apoptosis and inhibits proliferation in cells with FLT3-ITD mutation. Artificial trans-membrane peptides activating SHP-1 can reverse FLT3 associated aberrant signaling, thus providing a new potential strategy to target FLT3-ITD in AML. The possible mechanism is showed in figure G. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Elderly pts with R/R AML not eligible for cytotoxic therapy have limited therapeutic options, and dismal outcomes with available therapies. In preclinical studies, inhibition of BCL-2 and MDM2 with Ven and Idasa, respectively, has demonstrated potent synergistic apoptotic activity. In this ongoing, open-label, Phase Ib study, the safety, tolerability, and preliminary efficacy of Ven+cobimetinib (Arm A) and Ven+Idasa (Arm B) is being assessed in R/R AML (NCT02670044). Initial analysis indicated a tolerable safety profile for Ven+Idasa. Here, we present updated safety and efficacy results from Arm B. Methods: Pts (≥60 years) with R/R AML or secondary AML, previously treated for an antecedent hematologic disease but treatment naïve for AML, and ineligible for cytotoxic therapy/allogeneic stem cell transplant were enrolled. The maximum tolerated dose of Ven+Idasa was determined by two-dimensional dose escalation. Pts received Ven orally (PO) daily (400 or 600mg) + Idasa PO daily on Days (D) 1-5 (150mg, 200mg, or 400mg) in 28-day cycles. Responses were assessed according to revised International Working Group Response Criteria 2003. Pharmacokinetic (PK) analyses were performed on plasma samples on Cycles (C) 1 and 2, D1 and 5, and C4, D1. Exploratory assessments included minimal residual disease (MRD), assayed centrally at Covance Laboratories using 8-color flow cytometry. Data cut-off was June 21, 2019. Results: At data cut-off, 49 pts were treated with Ven+Idasa. Median age was 72 years (range 62-93); Eastern Cooperative Oncology Group performance status 0-1: 84%; refractory AML: 57%; relapsed AML: 33%; and secondary previously untreated AML: 10%; Intermediate-I or Intermediate-II European Leukemia Net (ELN) risk classification: 66%; adverse ELN classification: 30%; de novo (49%) versus secondary (51%) AML; and median prior lines of treatment: 1 (range 1-4). Most common adverse events (AEs; any grade) irrespective of attribution were diarrhea (90%) and nausea (78%); the most common grade ≥3 AEs were febrile neutropenia (45%), neutropenia (27%), and thrombocytopenia (25%; Table 1). Laboratory tumor lysis syndrome occurred in 3 pts; none resulted in treatment discontinuation. Ven and Idasa treatment discontinuation due to AEs were noted in 18% and 20%, respectively, most commonly due to infections. 30- and 60-day mortality rates were 6% and 17%, respectively. No apparent PK drug-drug interaction was found between Ven and Idasa; overlap in Ven and Idasa exposure was substantial over the doses tested. Anti-leukemic response rate (complete response [CR] + CR with incomplete platelet count recovery [CRp] + CR with incomplete blood count recovery [CRi] + partial response [PR] + morphologic leukemia-free state [MLFS]) across all dose levels was 41% (Table 2). Across the two Ven 600mg cohorts being considered for the recommended Phase II dose (RP2D), the anti-leukemic response rate was 50% (CR+CRp+CRi rate 29%). Median time to CR+CRp+CRi+PR was 1.4 months (range 1-3), with a median response (CR+CRp+CRi) duration of 4.9 months (range 0.6-9.7). Median overall survival in all pts and in the Ven 600mg cohorts was 4.4 months and 5.7 months, respectively, with a median follow-up of 3.4 months (range 0.03-18). Individual pt responses are shown in Figure 1. MRD negativity (