Survival In Acute Myeloid Leukemia Patients Research Articles

Suv39h1 Represses the Progression of MLL-Rearranged Myeloid Leukemia Via Hoxb13

Acute myeloid leukemia (AML) is the most frequent and heterogeneous malignancy in adult leukemic patients. Genome-wide analyses revealed that genes involved in epigenetic modifications are among the most often re-occurring mutations in AML, suggesting a crucial role of epigenetic regulation in leukemogenesis and leukemia relapse. As a mammalian lysine methyltransferase, SUV39H1 catalyzes di- and tri-methylation of histone 3 lysine 9, and is the predominant H3K9 methyltransferase expressed in hematopoietic stem cells (HSCs). Previous studies have shown that in MLL-rearranged leukemic cells, the normal localization of Suv39h1 and Sirt1 was interrupted due to the DNA binding of Dot1L to DNA. However, the biological role of SUV39H1 in MLL-rearranged leukemia remains unexplored.In this study, we investigated the role and the underlying mechanism of Suv39h1 during leukemia progression. By analyzing the clinical databases, we found a significantly reduced expression of SUV39H1 in AML cells in comparison with normal bone marrow (BM) cells. More importantly, we found that low expression of SUV39H1 predicts poorer survival in AML patients. In MLL-fusion induced AML mouse models (MLL-AF9/MA9 and MLL-NRIP3/MN3), Suv39h1 also exhibited lower expression in leukemia stem cells (LSCs, defined as c-Kit+ or Lin-Sca1-IL-7R-c-Kit+CD34+CD16/32+ L-GMP cells) when compared with normal HSPCs. These data suggest a potential role of SUV39H1 in leukemic progression and/or maintenance.To explore if Suv39h1 functions as a tumor suppressor in MLL-fusion driven leukemogenesis, we overexpressed Suv39h1 in MA9 BM AML cells. Western blotting analysis confirmed the overexpression of Suv39h1 with a moderate increase in global H3K9me3 levels in Suv39h1-overexpressed (SUV-OE) MA9 AML cells. Interestingly, Suv39h1 overexpression prolonged the survival of recipient AML mice in both secondary and tertiary transplantation groups. Both the frequency and the absolute number of phenotypic LSCs in BM and SP were significantly reduced in SUV-OE groups as manifested by flow cytometry. Furthermore, limiting dilution assays revealed a significant six-fold decrease of functional LSCs in SUV-OE AML cells (1/314 LSCs in SUV-OE AML cells vs 1/56 in controls). Cell cycle analysis of control and SUV-OE LSCs from BM revealed a significantly decreased proportion of SUV-OE cells in the S/G2/M phase concordant by an increased proportion of G0/G1 phases when compared with control cells. In contrast, a similar apoptotic ratio of L-GMPs in BM was observed between control and SUV-OE groups. Taken together, these data demonstrated that overexpressing Suv39h1 in AML cells reduces the frequency of functional LSCs by suppression its proliferation.To explore the underlying mechanisms, gene expression profiles were assessed by RNA-Seq of SUV-OE and control mouse AML c-Kit+ cells. A total of 69 genes were differentially expressed with fold change ≥ 4. Among these genes, Hoxb13 was of particular interesting since it was reported to be recurrently mutated in several types of cancers including leukemia. ChIP-qPCR revealed a two-fold increase of H3K9m3 distribution at the promoter of Hoxb13 in SUV-OE groups, indicating Hoxb13 may be a direct downstream target of Suv39h1. Restoring the expression of Hoxb13 in SUV-OE AML cells diminished the effect of SUV-OE-mediated prolonged survival of SUV-OE AML mice. Interestingly, overexpression of Hoxb13 alone in MA9 cells had no significant effect on the survival of MA9 AML mice, indicating that Hoxb13 is a downstream effector of Suv39h1, rather than MA9, and Suv39h1 itself is a downstream mediator of MA9.To summarize, we here for the first time, demonstrate that Suv39h1 is significantly down-regulated in AMLs and could function as a tumor suppressor in MLL-rearranged leukemia by epigenetically inhibiting the Hoxb13 expression. The molecular mechanism mediated by Suv39h1-Hoxb13 axis in tumor suppression could potentially provide us novel therapeutic strategies for MLL-rearranged leukemia.YJ.C, YP.C and HD.G contributed equally to this work.Corresponding authors: WP.Y and MJ.X. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Identification of Novel Lncrnas That Predict Survival in AML Patients and Modulate Leukemic Cells

Long noncoding RNAs (lncRNAs) have complex, mainly chromatin-associated functions and their expression is highly coordinated and cell-type specific. Based on their tight regulation in normal differentiation, we set out to investigate whether lncRNAs are dysregulated in diseases where differentiation is impaired, such as in acute myeloid leukemia (AML). To identify lncRNAs that are essential for both normal hematopoiesis as well as AML maintenance, we sequenced the long polyA- and non-polyA-tagged transcriptome from successive stages of human myelopoiesis (myeloblasts, promyelocytes, metamyelocytes, and neutrophils) isolated from bone marrow of healthy donors (n=3). Applying a high-dimensional data portraying approach (OposSOM, Löffler-Wirth et al., BMC Bioinformatics, 2015), we identified functional expression modules of lncRNAs that are either positively or negatively associated with myeloid lineage commitment in our dataset. Seven out of the top15 differentiation-associated lncRNAs exhibit significant prognostic relevance in overall and event-free survival analyses of independent AML patient datasets and improve the predictive power of the current prognosis standards (cytogenetic risk/age/TP53-status). In particular, a combination of 3 transcripts, PROMYS (Promoter of Myelopoiesis, annotated as uncharacterized ncRNA LOC107985167), ANTAMY (Antagonist of Myelopoiesis, uncharacterized ncRNA LOC101927745) and LINC00677, outperformed the recently reported prognostic benefit of the LSC17high score (Ng et al, Nature, 2016) by a factor of Ø 22.7 based on concordance index score increase (Ø 4.8% vs. 0.21%).All three lncRNAs are highly conserved, expressed in 10 tested human AML cell lines as well as significantly differentially expressed in distinct cytogenetic patient subgroups of The Cancer Genome Atlas (TCGA) LAML cohort (n=171). PROMYS is downregulated in t(15;17) and t(8;21) cases, supporting its strong association with worse OS in the TCGA-LAML dataset (p=0.0001). In contrast, ANTAMY shows high expression in AML with t(8;21), and LINC00677 in NPM1+/FLT3- mutated AML patient samples with normal karyotype (CN-AML) and in core Binding factor (CBF) AMLs. Accordingly, high expression levels of both lncRNAs associate with a significantly better OS in the TCGA LAML dataset (p=0.01 and 0.02, respectively).To investigate their function in vitro, we knocked out each lncRNA individually in the human OCI/AML-5 AML cell line using CRISPR/Cas9. Loss of ANTAMY impaired proliferation (p=0.04) and increased both monocytic differentiation upon treatment with 2-0-tetradecanoylphorbol-13-acetate (TPA) (p=0.0001) and granulocytic differentiation with all-trans retinoic acid (ATRA) (p=0.0002) compared to the empty vector control. Loss of LINC00677 in OCI/AML-5 cells specifically increased granulocytic differentiation through ATRA (p=0.0002). In contrast, inactivation of PROMYS led to reduced differentiation induced by ATRA (p=0.00004) and TPA (p=0.002). Furthermore, we found that PROMYS is involved in the regulation of the Macrophage colony-stimulating factor 1 (CSF1), which is deregulated in ATRA- and TPA-induced differentiation in PROMYS knockout but not in control cells (p<0.002 and <0.00002, respectively), explaining its negative impact on differentiation. Through screening of human myelopoiesis, we identified three unexplored lncRNAs: LINC00677, PROMYS, and ANTAMY, which play a role in myeloid differentiation and have an impact on patient prognosis. Our in vitro findings confirm that ANTAMY, LINC00677, and PROMYS are active modulators of leukemic cells, which influence their proliferation, morphology, myeloid marker expression as well as apoptosis rate. These transcripts and their interaction partners add an additional layer of regulation to the understanding of differentiation and might represent previously unknown vulnerabilities of AML cells, which warrants their further investigation in vivo. DisclosuresNo relevant conflicts of interest to declare.

Longer Distance to a Specialized Treatment Center Does Not Adversely Affect Access to Treatment or Outcome in Acute Myeloid Leukemia: A National Population-Based Cohort Study

BACKGROUNDTreatment of acute myeloid leukemia (AML) is widely centralized at specialized centers. Longer distances to a specialized treatment facility may affect patients' access to curative-intended treatment and ultimately survival. Few studies have focused on the potential distance decay association in hematological cancers and limitations include small sample size and lack of individual-level socioeconomic, clinical, and treatment information.AIMWe designed a large national population-based cohort study of all AML patients diagnosed in Denmark between 2000-2014 (followup ending 2017) to investigate the effect of distance on treatment intensity and outcome considering individual-level clinical and socioeconomic factors.METHODSDemographics, clinical, and outcome data were obtained from the Danish National Acute Leukemia Registry (DNLR). Socioeconomic information was retrieved from registries at Statistics Denmark (Figure 1).Distance to specialized treatment centres was calculated using shortest route (Google Maps) from city center of habitation and categorized into groups.We investigated effects of distance to nearest specialized treatment center on chance of receiving intensive chemotherapy using logistic regression analysis (odds ratios; ORs). In intensive therapy patients, we calculated chance of complete remission (CR [ORs]) and in allogeneic transplantation (HSCT) candidates; we estimated chance of HSCT (ORs). Overall survival was calculated for all patients and in intensive therapy patients only using cox regression (Hazard Ratios; HRs).All results were adjusted for sex, age, and individual-level socioeconomic (ethnicity, income, education, and occupation) and disease-related factors (WBC, secondary or therapy-related AML, cytogenetics, performance status, and comorbidity). Results were given crude and adjusted with 95% confidence intervals (CI), and stratified by age (<65 years; ≥65 years).RESULTSOf 2992 patients (median age 68.5 years), 53% received intensive chemotherapy, and 71% (n=1045) achieved a CR. The median distance to a specialized treatment center was 40 km (interquartile range 10-77 km). Patients living furthest from a specialized center had lower income and lower educational level.No negative impact of distance to specialized treatment center was seen on chance of receiving intensive therapy (adjusted ORs: <10 km 1.0, 10-25 km 1.10 (CI=0.69-1.73), 25-50 km 1.10 (CI=0.73-1.66), 50-100 km 1.33 (CI=0.90-1.97), and >100 km aOR 1.40 (CI=0.90-2.16)).In intensive therapy patients (n=1588), distance to specialized treatment center did not affect time from diagnosis to chemotherapy initiation or chance of achieving a CR.In HSCT candidates (866 patients ≤70 years with non-favorable cytogenetics achieving CR), longer distance to transplant center was not negatively associated with chance of HSCT (adjusted ORs: distance<50 km 1.0, 50-200 km 1,27 (CI=0.56-2.90), and >200 km 0.77 (CI=0.34-1.74)).The median follow up time was 514.5 days. Figure 2 shows crude survival by distance to specialized treatment center overall and in selected subgroups. Overall survival was not affected by distance to specialized treatment center (adjusted HRs: distance <10 km 1.0, 10-25 km 0.98 (CI=0.84-1.16), 25-50 km 1.01 (CI=0.88-1.18), 50-100 km 1.01 (CI=0.88-1.16), and >100 km 0.99 (CI=0.85-1.16)). Similar results were found in intensive patients only.Stratifying by age or analyzing distance as a continuous variable did not change the overall interpretation of results.CONCLUSIONIn a population-based national setting, distance to specialized treatment facilities offering remission-induction chemotherapy and HSCT, does not affect access to care, treatment outcome, or survival in AML patients. These findings support that the current centralization of specialized treatment does not negatively affects AML outcomes in a universal health-care system. DisclosuresNo relevant conflicts of interest to declare.

A Potential Mechanism for the Antioxidant Compound Kushen Injection Targeting the ROS Signaling Pathway in Acute Myeloid Leukemia

AbstractIt is a challenge for treatment of acute myeloid leukemia (AML) in clinic. The increase in the levels of reactive oxygen species (ROS) in AML patients has been previously described; thus, it is important to regulate ROS levels in AML.In this study, we found that intracellular ROS levels in AML cells were decreased, the total antioxidant capacity (T-AOC) and glutathione (GSH) contents were increased and xanthine oxidase (XOD) vitality was decreased when treated with the compound kushen injection (CKI). This shows that CKI inhibited the proliferation of AML cell lines and patient cells and enhanced the cytotoxicity of AML cells, which has few toxic effects on haematopoietic stem cells (HSCs) and T cells. At the single-cell level, individual AML cells died gradually by CKI treatment on optofluidic chips. CKI could promote apoptosis and arrest cell cycle at G1/G0 phase in U937 cells.Furthermore, higher peroxiredoxin-3 (Prdx3) expression levels were found in CKI-treated U937 cells through quantitative proteomics detection. Mechanically, the expression of Prdx3 and peroxiredoxin-2 (Prdx2) was up-regulated in CKI-treated AML cells, while thioredoxin 1 (Trx1) was reduced. Laser confocal microscopy showed that the Prdx2 and Trx1 proteins could be co-localized by CKI treatment.In vivo, in the CKI-treated group, survival was longer in an AML patient-derived xenograft model in B-NSG mice. The disease was partially alleviated by decreased CD45+ immunophenotyping in peripheral blood and bone marrow smear analysis. After CKI injection, the T-AOC vitality, GSH content and CAT activity increased and the concentration of H2O2decreased in mouse plasma. A bone marrow biopsy and immunohistochemistry analysis showed that Prdx3 and Prdx2 expression was increased, while Trx1 expression was decreased.In a conclusion, we provided a model for the anti-leukaemic effects of CKI. CKI decreased intracellular ROS levels by up-regulating the expression of Prdx2 in the cytoplasm and Prdx3 in the mitochondria and down-regulating Trx1 expression, which maintained the intracellular REDOX and further inhibited AML cell proliferation.Therefore, antioxidant CKI is a promising drug for the treatment of AML in the clinic. We aimed to explore the therapeutic efficacy of low toxicity natural antioxidants against AML by targeting ROS pathways and providing new strategies to improve survival in AML patients. DisclosuresNo relevant conflicts of interest to declare.

A Novel Role for Bst2 and E-Selectin in IFNg-Stimulated HSC Niche Relocalization

Prolonged exposure to proinflammatory conditions, such as during chronic infection, can lead to the development of bone marrow failure syndromes including aplastic anemia. Indeed, we have previously shown that chronic infection activates hematopoietic stem cells (HSCs), ultimately leading to bone marrow suppression due to an IFNgamma (IFNγ)-dependent depletion of HSCs. Defining the mechanisms behind inflammation mediated HSC loss may provide important insight into how inflammation affects hematopoiesis in many clinical conditions including infection, ageing, and myelodysplastic syndrome.HSC quiescence is maintained in large part through interactions with the surrounding cells of the bone marrow niche. In particular, HSCs closely colocalize with perivascular stromal cells that secrete high levels of the chemokine Cxcl12 and other hematopoietic maintenance factors such as angiopoietin, IL7, and stem cell factor. We hypothesized that IFNγ-induced activation may affect the positioning of HSCs relative to supporting cells within the bone marrow microenvironment. We used 3-D intravital imaging to assess the localization of HSCs relative to Cxcl12-abundant reticulocytes (CAR cells). When CMTMR-stained HSCs were transplanted into Cxcl12-GFP mice we found that IFNγ treatment led to a distancing of HSCs away from quiescence-enforcing CAR cells. In contrast, TNF-alpha treatment, which does not induce HSC proliferation at the concentration used, resulted in no such relocalization. Furthermore, no displacement occurred in response to IFNγ treatment when Ifngr1-deficient HSCs were transplanted, suggesting a cell-autonomous mechanism of relocalization.RNA expression analysis and chemotaxis assays showed that IFNγ-treated HSCs are not impaired in their ability to respond to Cxcl12 signaling, indicating that a change in Cxcl12-Cxcr4 interactions does not account for the movement of HSCs away from CAR cells. To further assess HSC-specific changes that may account for IFNγ-dependent relocalization, we performed gene expression analysis of HSCs from control or IFNγ-treated mice. We saw no change in the expression of common HSCs receptors that are thought to play a role in maintaining HSC quiescence, such as cKit, Cdh2, Mpl, Itgb1, Itbg2, Itga4, and Itga1. However, the surface protein, Bst2, also known as tetherin, was significantly upregulated in HSCs upon IFNγ stimulation. This was confirmed at both the RNA and protein level. A prior report identified Bst2 as a noncanonical E-selectin ligand. This finding is particularly interesting given that HSCs found in close proximity to E-selectin-expressing endothelial cells exist in an activated, proliferative state. Furthermore, the interaction between HSCs and E-selectin+ endothelial cells was previously noted to be independent of canonical E-selectin ligands such as Psgl1, CD44, or the Lewis family antigens. Thus we hypothesized that IFNγ-induced upregulation of surface Bst2 may facilitate proliferation by promoting HSC binding to E-selectin. Indeed, in vitro E-selectin binding assays showed that IFNγ treatment increased binding of progenitor cells to E-selectin, but not to the closely related adhesin, P-selectin.Inflammation plays an important role in the development of many cancers including acute myeloid leukemia (AML), leading us to investigate if Bst2 upregulation is an important factor in AML biology. Indeed, we found that increased levels of Bst2 are associated with poor survival in AML patients. Further, murine studies have shown that leukemic blasts have greatly increased E-selectin binding capacity following transformation. Using two independent AML cell lines, we found that IFNγ treatment upregulates Bst2 expression and increases E-selectin binding capacity. These findings suggest a mechanism by which inflammation could drive cell proliferation in AML.In summary, we have uncovered a potential new mechanism for IFNγ-induced HSC activation, whereby HSCs are relocalized in a Bst2-dependent manner from the quiescence-enforcing CAR niche to an active E-selectin-positive niche. Furthermore, we demonstrate that IFNγ-mediated Bst2 expression also occurs in AML, suggesting a mechanistic link between inflammation and AML progression. This study may open up new potential therapeutic avenues for the treatment of patients with chronic infection, inflammatory diseases, and cancer. DisclosuresNo relevant conflicts of interest to declare.

Novel Therapeutic Targeting of UHRF1 Restores 5'-Azacytidine Response in Resistant Acute Myeloid Leukemia

DNA hypermethylation plays a pivotal role in the pathogenesis of acute myeloid leukemia (AML). Most of the recurrent driver mutations and chromosomal translocations in AML involve genes encoding chromatin modifiers and DNA methylation relevant enzymes. Hypo-methylating drugs such as 5-Azacytidine (AZA) that target DNMTs prolong overall survival in AML patients. However, their long term treatments lead to emergence of acquired therapy resistance mostly through unknown mechanisms and hence there is an urgent need for alternate therapeutics to address AZA resistance in AML patients. Recently, it has been shown that AZA resistant leukemic cells are relatively quiescent with higher expression of many components of DNA methylation machinery that also includes UHRF1 (ubiquitin-like with PHD and ring finger domains 1). UHRF1 is a key epigenetic modulator that regulates DNA methylation and gene expression. It is a multi-domain nuclear protein with an SRA (SET-and-RING-associated) domain to recognize hemi-methylated DNA immediately after replication. It plays a crucial role in the maintenance of DNA methylation by recruiting DNMT1 to replication sites and facilitates methylation on newly synthesized DNA strand. UHRF1 is frequently overexpressed in multiple human neoplasms including AML and in the absence of UHRF1, hematopoietic stem cells undergo erythroid-biased differentiation at the expense of self-renewal capacity. Despite UHRF1 being key a therapeutic target against AML, specific, and cell-permeable inhibitors of UHRF1 have not been identified yet.In this study, we hypothesized that targeting UHRF1 using novel small molecule inhibitor will interfere with DNMT1-dependent DNA methylation at newly synthesized DNA strand, which may further synergize with antiproliferative effect of classical DNMT inhibitors in AML cells. In this study, we used in silico strategy to discover novel putative UHRF1 inhibitors by screening NCI compound database. For in vitro validation, we have first purified the SRA domain of UHRF1 followed by analysis of total DNA methylation levels using 5'-methyl cytosine (5mC) dot blot in the presence of each inhibitor. After a series of stringent in vitro and cell based assays we have identified lead compound 20 (C20) as a potent UHRF1 inhibitor which suppresses DNA methylation without affecting DNMTs in leukemic cells. Specificity of C20 against SRA domain was further established by isothermal titration calorimetry (ITC). We next found that C20 treatment significantly decreased UHRF1 and DNMT1 foci formation in the nucleus of mouse embryonic fibroblast and stem cells. Based on the its critical role in DNA methylation and enhanced expression in resistant cells, we assumed that AZA resistance in AML may be mediated by UHRF1 and C20 might restore AZA sensitivity by attenuating enhanced UHRF1 activity. To validate this, we pretreated AZA resistant leukemic cells (HL60R) with suboptimal dose of C20 followed by AZA treatment. Interestingly, we found a synergistic increase in antiproliferative effect by flow cytometry and colony formation assay. By analyzing the surface expression of myeloid differentiation markers, we found that C20 treatment promotes differentiation and decreases quiescent leukemic cell population. In conclusion, we report a novel UHRF1 inhibitor as a sensitizer of resistant AML cells towards AZA treatment potentially by promoting differentiation, suggesting a novel combination approach for future clinical evaluations. DisclosuresNo relevant conflicts of interest to declare.

Hematopoeitic Cell Transplant - Comorbidity Index (HCT-CI) Score Is a Useful Tool for Predicting Induction Mortality and Overall Survival in Newly Diagnosed Acute Myeloid Leukemia Patients

Intro:Assessment of induction mortality risk in acute myeloid leukemia (AML) patients is challenging. Hematopoietic cell transplant comorbidity index (HCT-CI) score was designed for risk assessment of patients undergoing an allogeneic transplant. However, it is often used in clinical practice to assess induction mortality risk in AML patients. We analyzed the predictive power of the score to assess induction mortality risk as well as overall survival in AML patients at our institution.Methods:Between Jan 2009 and March 2013, 101 consecutive newly diagnosed AML patients treated at our institution were included for analysis. All patients received bolus HiDAC (3gm/m2) on day 1-5 and high dose mitoxantrone (80mg/m2) on day 2. The median age was 65 years (range18-90). The total number of patients in the age group <60, 60-69 and ≥70 were 30(29.7%), 35(34.6%) and 36(35.6%) respectively. HCT-CI scores were calculated before induction chemotherapy. Kaplan Meir curve was used to calculate OS and PFS. 4-week induction mortality was calculated from the date of the induction chemotherapy. The patient cohort was divided into those with HCT-CI scores of > or ≤3.Results:The number of patients with the HCT-CI score of 0,1,2 and 3 were 8,13,17 and 13 respectively. 50% of our patients (51 patients) had a score of 4 or more on the HCT-CI index. The 4 and 8-week mortality in our cohort was 3/101 (2.9%) and 7/99 (7%) respectively. 4-week induction mortality in <60, 60-69 and ≥70 years of age groups were 0/30(0%), 1/35(2.8%) and 2/36(5.5%) respectively. Similarly, in above age groups, 8-week induction mortality were 0/29(0%), 2/34(5.8%) and 5/36(13.8%) respectively. The median OS in patients with an HCT-CI score ≤3 was 87 months versus 11.5 months in those with a score >3 (log-rank, p=0.0046, Fig1). 2-yr NRM rate for patients with HCT-CI score ≤3 and >3 was 47% and 71%, respectively (p=0.01, Fig2). Hazard ratio for overall mortality in patients with HCT-CI score >3 is 1.793 with p=0.023, 95% CI = (1.083 - 2.971). HR for 8-week mortality in patients with an HCT-CI score >3 is 2.035 (p=0.412), although not statistically significant due to a low number of events. However, by Cox multivariable regression analysis HCT-CI retained a significant impact on 8-week mortality (HR=1.24, p= 0.03) and overall survival (HR=1.144, p= 0.006).Conclusion:An HCT-CI score is a valuable tool in the assessment of the induction mortality in newly diagnosed AML patients and can be used for risk assessment of overall mortality. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Population Pharmacokinetic/Pharmacodynamic Evaluation of the Relationship between Glasdegib Treatment/ Exposure and Overall Survival in AML Patients

Introduction: Glasdegib is an inhibitor of the Hedgehog signaling pathway through binding to its target, Smoothened (SMO). B1371003 was a Phase 1b/2 study evaluating glasdegib in combination with chemotherapy in patients with acute myeloid leukemia (AML). In one part of the Phase 2 portion of B1371003, patients who were not candidates for more standard chemotherapy were randomized 2:1 to receive low-dose cytarabine (LDAC) with glasdegib or LDAC alone. The primary endpoint was overall survival (OS). A Cox regression analysis for overall survival (OS) was conducted, and median OS for AML patients treated with glasdegib plus LDAC was 8.3 months compared to 4.3 months with LDAC alone (hazard ratio [HR] 0.463). This analysis aimed to assess the potential relationship between glasdegib exposure and OS (exposure-response [ER]) and to evaluate the effect on OS of moderators/covariates, including treatment-response (TR) for glasdegib + LDAC vs. LDAC alone.Methods: The analyses included data from the Phase 2 non-intensive AML patients. Glasdegib was administered continuously at 100 mg once daily (QD) orally in 28-day cycles. LDAC was administered subcutaneously at 20 mg twice daily on Days 1-10 of each 28-day cycle. Glasdegib exposure from the glasdegib + LDAC treated arm was estimated utilizing a population pharmacokinetics (PK) model. Survival was assessed at every visit then every month for the first 2 months after discontinuation from study treatment and thereafter every 2 months until death or termination of the study.Parametric time-to-event survival models were used to assess the relationship between OS, treatment arm, and glasdegib exposure. Baseline characteristics (such as age, sex, body weight, and white blood cell count) and other prognostic factors (such as disease status and treatment arm) were evaluated as potential predictors of OS by forward inclusion and backward elimination. R statistical programming (version 3.2.2) and NONMEM software (version 7.3.0) were used for the analysis.Results: Glasdegib PK was adequately characterized by a 2-compartment model with first order absorption kinetics. Demographic and baseline characteristics such as age, sex, body weight, and renal and hepatic function were either non-significant or did not have a clinically relevant impact on glasdegib PK. The TR analysis included all Phase 2 AML patients with available OS data who were randomized to receive LDAC with (n=78) or without (n=38) glasdegib. Glasdegib-treated patients with at least one PK observation (n=75) were evaluated in the ER analysis. Exponential, Weibull, and log-logistic parametric distributions were explored, and the exponential distribution was selected as the model that best described the observed OS data (Figure). In the TR analysis, addition of glasdegib to LDAC resulted in longer OS with HR of 0.42 (95% confidence interval: 0.28-0.66). No ER relationship was identified in the analysis between glasdegib plasma exposures at 100 mg QD and OS. No other baseline characteristics were identified as potential predictors of survival in either the TR or ER analyses.Conclusions: Addition of glasdegib treatment to LDAC chemotherapy in AML patients who are not suitable for intensive chemotherapy significantly prolonged OS compared to LDAC chemotherapy alone. Across the range of plasma exposures for glasdegib 100-mg QD treatment, no ER relationship was identified. No other covariates were found to be predictors of survival, therefore supporting the broad use of glasdegib as an addition to chemotherapy in AML patients. [Display omitted] DisclosuresLin:Pfizer: Employment. Shaik:Pfizer Inc: Employment, Equity Ownership. Chan:Pfizer: Employment, Equity Ownership. Cortes:Daiichi Sankyo: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Arog: Research Funding. Ruiz-Garcia:Pfizer: Employment, Equity Ownership.

Survival of AML Patients Relapsing after Hematopoetic Stem Cell Transplantation: A Single Center Experience

Although it is presumed to be a curative strategy for intermediate and high risk acute myeloid leukemia (AML), many patients relapse after allogeneic hematopoietic stem cell transplantation. This prompt us to examine the ways to improve the outcomes. We retrospectively evaluated 76 AML patients who were transplanted between 2007-2017 years in our clinic. We tried to identify the factors associated with posttransplant relapse, postrelapse survival and if there was a survival benefit of pretransplant consolidation and minimal residual disease (MRD) negativity. We examined the effect of the acute-chronic graft versus host disease (GVHD) and salvage therapy after the posttransplant relapses. The mean age of the patients was 44.6±1.21 years (ranges 21-67). 42.1% were females and 57.9% were males. 43.3% of the patients were in complete remission (CR) MRD positive state before the transplantation whereas 35.5% were in CR MRD negative and 3.9% were in progressive disease state. In 13 patients who were in CR state, the MRD status were not known. 11 (14.5%) patients were considered as in favorable risk, 52 (68.4%) in intermediate risk and 13 (17.1%) in unfavorable risk with respect to cytogenetic analysis before the transplantation. The donors were HLA compatible relatives (77.6%), HLA compatible non-relatives (10.5%), haploidentical people (9.2%), one HLA incompatible relative (1.3%) and one HLA incompatible nonrelative (1.3%). 74 bone marrow transplantations (97.4%) were allogeneic and the remaining two (2.6%) were autologous. Myeloablative conditioning regimen was applied to 57 patients (75%) and 19 patients got (25%) reduced intensity conditioning regimen. GVHD developed in 51.3% after transplantation and 61.5% of these were chronic extensive. Relapse occurred in 27 patients (35.5%), hematological relapse being the most common (31.6%). The median time for the development of relapse was found to be 5.5 months (range: 1.5-37). The overall probability for the development of a relapse was found to be 48.7% (95% CI: 40.9-56.5)(Figure1A). 23 patients (30.3%) died during the study period with a median survival of 9.6 months (range: 1.6-45). In the studied population the overall survival probability was found to be 52.8% (95% CI: 45.4 - 60.2) [Figure 1B]. 36.4%, 28.8% and 30.8% of the patients with favorable, intermediate and unfavorable cytogenetic status died respectively during the study period. The comparison of the survival probability of the patients with favorable, intermediate and unfavorable cytogenetic status was depicted in Figure 2. The overall survival probability of the patients with favorable, intermediate and unfavorable cytogenetic status were 46.6% (95% CI: 26.2-66.9), 54.6% (95 % CI: 45.9 - 63.2) and 36.9 % (95% CI: 25.4 - 48.5) respectively (p=0.807).MRD status of 60 patients were known. At the end of the study period 75.8% of the CR MRD positive and 70.4% of the CR MRD negative patients remained alive. The comparison of the survival of patients in CR with respect to MRD status is shown in Figure 3. The overall survival probability of CR MRD positive patients was 56.3 % (95% CI: 45.3 - 67.3) and this rate was 52.5 % (95% CI: 40.8 - 64.3) in MRD negative patients (p=0.770) [Figure 3].Patients who developed GVHD had similar overall survival probability with the patients who did not developed the disease; 47.0% vs 57.2%, p=0.115 (Figure 4A). Even the patients with chronic extensive GVHD had similar overall survival rates with the patients who had none or acute GVHD; 49.3 % vs 58.2%, p=0.27 (Figure 4B).66.7% of the patients with a progressive disease before the transplantation died during the study period and this rate was 27.1% in the patients with CR (p=0.005).In conclusion the overall survival rate of the transplanted AML patients was 52.8% in the study group. The overall survival did not seem to be affected by pre-transplant MRD status, cytogenetic risk factors and administration of consolidation therapy. The only patients who had significantly worse results were the ones who had progressive disease before the transplantation. From this point it would be logical to make transplantation whenever the patient is in first CR regardless of the MRD status and a matched donor is found so that the toxic effects of the consolidation chemotherapy may be prevented. DisclosuresNo relevant conflicts of interest to declare.

Insulin-like Growth Factor Binding Protein 7 Activates the Retinoid Acid Differentiation Pathway in Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired transcriptional differentiation programs. Despite important advances in AML therapy, the five-year overall survival rate of AML patients remains a disappointing 30-40%. This poor prognosis is mainly caused by survival of chemotherapy resistant leukemic cells, named leukemic stem cells (LSC), re-initiating relapse. However, for AML patients with PML-RARA positive acute promyelocytic leukemia (APL), treatment with all trans retinoic acid (ATRA) results in cure rates of >90%. Upon ATRA treatment, APL cells can restore transcription leading to granulocytic differentiation, and in combination with arsenic trioxide APL cells go into apoptosis. While the success of ATRA treatment has been demonstrated for APL patients, so far it has not proved effective for non-APL AML patients.Previously, we demonstrated that insulin-like growth factor binding protein 7 (IGFBP7), a negative regulator of IGF1 receptor (IGF1R) activity, induces apoptosis of AML cells and sensitizes AML cells to chemotherapy-induced cell death. Since it has been shown that IGF1R inhibitors can eliminate therapy-resistant cells by modifying their chromatin state, we hypothesized that IGFBP7 may also have the capacity to modify an epigenetic state and unlock the ATRA-driven differentiation response. To this end, we generated APL cell lines with down- or upregulated IGFBP7 levels and demonstrated that knockdown of IGFBP7 in NB4 cells blocked ATRA-induced differentiation, whereas overexpression of IGFBP7 led to an 8-fold increase in differentiation in the presence of low concentrations of ATRA, together suggesting a role for IGFBP7 in ATRA-induced differentiation in APL cells.Strategies to increase efficacy of ATRA-based therapy might also improve treatment outcomes for non-APL AML, and therefore we investigated the potential of IGFBP7 to induce susceptibility for ATRA-driven differentiation in this group. ATRA and IGFBP7 treatment of non-APL AML cell lines and primary AML cells derived from patients at diagnosis demonstrated an enhanced efficiency of the combination therapy to induce differentiation of myeloid CD45dimCD33+ AML cells (2.5-fold increase in CD11b-expression) and/or to reduce viability of AML CD45dim cells as compared to single treatments (from 30-39% to 70% reduction upon IGFBP7, ATRA or combination therapy, respectively), in 50% of tested primary AML samples, while this combination therapy did not influence normal hematopoietic cell survival. Remarkably, ATRA-IGFBP7 treatment diminished the in vivo engraftment potential of primary AML cells as compared to treatment with either drug alone in NSG mice (from 28% and 52% to 17% engraftment upon ATRA, IGFBP7 or combination therapy, respectively). Re-transplantation of human AML derived from first transplanted mice into secondary recipients demonstrated that the ATRA-IGFBP7 combination treatment also eliminated LSC more effectively (1.4-fold reduction). Together these data suggest that IGFBP7 enhances sensitivity of AML (stem) cells to ATRA, and is able to induce a transcriptional program sensitizing AML cells for ATRA-induced differentiation and cell death.To identify factors responsible for IGFBP7-induced ATRA sensitivity, we performed gene expression profiling of primary AML samples treated with IGFBP7, and identified growth factor independent protein 1 (GFI1) as one of the top down-regulated genes upon IGFBP7 stimulation. As overexpression of GFI1 in non-APL AML patient samples resulted in a >2.5-fold reduction in IGFBP7-induced susceptibility to ATRA-driven differentiation, low GFI1 expression is suggested to be associated with susceptibility to ATRA in AML cells.In conclusion, our results indicate that treatment of AML patient with a combination of ATRA and IGFBP7 might be successful in preventing relapse and improving AML patient survival, which has to be confirmed in further clinical studies. DisclosuresOssenkoppele:Karyopharm: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Roche: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Genmab: Research Funding; Celgene: Honoraria, Research Funding; Johnson & Johnson: Consultancy, Honoraria, Research Funding.

CD56 and CD11b Positivity with Low Smac/DIABLO Expression as Predictors of Chemoresistance in Acute Myeloid Leukaemia: Flow Cytometric Analysis

Background:Resistance to chemotherapy is a major obstacle to curing acute myeloid leukaemia (AML), and several antigens are claimed to play primary roles in this resistance.Purpose:The aim of this study was to evaluate the roles of CD56, CD11b and Smac/DIABLO gene expression levels as prognostic markers of the clinical outcome, response to chemotherapy and survival of AML patients.Materials and Methods:A cross-sectional observational study was conducted on 60 naïve-AML patients who received induction therapy with mitoxantrone and cytarabine combined with a high dose of cytarabine. The CD56,CD11b and Smac/DIABLO expression levels were assessed using flow cytometry at diagnosis and were analysed for correlation with the possible associated risk factors, response to chemotherapy, and median duration of disease-free survival (DFS) and overall survival (OS).Results:The overall results revealed that AML patients who exhibited positive expression for CD56 and CD11b had short median durations of DFS and OS.(P = 0.019, 0.006, 0.029 and 0.024, respectively). Additionally, low Smac/DIABLO expression had a negative impact on treatment outcome in terms of CR rate (p=0.012) and reduced DFS (p=0.000) and OS(p=0.000) values.Conclusions:CD56 and CD11b positivity and low Smac/DIABLO expression are important predictive factors for the occurrence of chemoresistance, in addition to other risk factors, among AML patients.

Acute Myeloid Leukemia and the Bone Marrow Niche—Take a Closer Look

The bone marrow is the home of hematopoiesis and is therefore a hotspot for the development of hematopoietic diseases. Complex interactions between the bone marrow microenvironment and hematopoietic stem cells must find a balance between proliferation, differentiation and homeostasis of the stem cell compartment. Changes in this tightly regulated network can provoke malignant transformation, leading to hematopoietic diseases. Here we focus on acute myeloid leukemia (AML), since this is the most frequent acute leukemia in adulthood with very poor overall survival rates and where relapse after chemotherapy continues to be a major challenge, driving demand for new therapeutic strategies. Current research is focusing on the identification of specific interactions between leukemic blasts and their niche components, which may be exploited as novel treatment targets along with induction chemotherapy. Significant progress has been gained over the last few years in the field of high-resolution imaging. Confocal ex vivo and intravital microscopy have revealed a detailed map of bone marrow structures and components; as well as identifying numerous alterations in the stem cell niche that correspond to disease progression. However, the underlying mechanisms are still not completely understood and due to the complexity, their elucidation remains a challenging. This review discusses the constitution of the AML niche in the bone marrow, the improvement in visualization of the complex three-dimensional niche structures and points out new therapeutic strategies to increase the overall survival of AML patients.

TMEM18: A Novel Prognostic Marker in Acute Myeloid Leukemia

Background: Certain nuclear envelope proteins are associated with important cancer cell characteristics, including migration and proliferation. Abnormal expression of and genetic changes in nuclear envelope proteins have been reported in acute myeloid leukemia (AML) patients. Transmembrane protein 18 (TMEM18), a nuclear envelope protein, is involved in neural stem cell migration and tumorigenicity. Methods: To examine the prognostic significance of TMEM18 in AML patients, we analyzed an AML cohort from The Cancer Genome Atlas (TCGA, n = 142). Results: Kaplan-Meier survival analysis revealed that TMEM18 overexpression was associated with a better AML prognosis with good discrimination (p = 0.019). Interestingly, this ability to predict the prognosis was significant in male AML patients, but not in female ones. C-index and area-under-the-curve analyses further supported this discriminative ability and multivariate analysis confirmed its prognostic significance (p = 0.00347). Correlation analysis revealed that TMEM18 had a statistically significant positive correlation with nuclear envelop protein 133 (NUP133), NUP35, NUP54, NUP62, and NUP88. Conclusion: Because the current AML prognostic factors do not take mRNA expression into consideration unlike other cancers, the development of mRNA-based prognostic factors would be beneficial for accurate prediction of the survival of AML patients. Therefore, TMEM18 gene is a potential biomarker for AML.

Clinical characteristics and prognosis of 34 cases of acute myeloid leukemia with FLT3 internal tandem duplication and MLL gene rearrangement

目的探讨同时伴FLT3-ITD突变及MLL基因异常的急性髓系白血病（AML）患者的临床特征及转归。方法回顾性分析34例同时伴FLT3-ITD突变及MLL基因异常的AML患者的临床资料，比较化疗、化疗加靶向药物治疗及allo-HSCT的疗效及影响因素。结果34例同时伴FLT3-ITD突变及MLL基因异常的AML患者占同期住院AML患者的2.02%。入院时WBC>30×109/L的患者占63.6%，其中WBC>50×109/L者占39.4%。FAB亚型中以M5比例最高，占35.3%，染色体核型异常者达63.6%，其中复杂异常占12.1%。34例患者中仅有FLT3-ITD及MLL基因异常（双基因异常）者11例（32.4%），具FLT3及MLL以外的1种及1种以上的基因异常（多基因异常）者23例（67.6%）。34例患者2个疗程完全缓解（CR）率为29.4%，7例（20.6%）化疗≥3个疗程后CR，CR患者的早期复发率为52.9%。WBC>50×109/L以及多基因异常的患者2个疗程CR率较低（7.7%、5.4%），其中具有3种以上基因异常的患者无一例CR。34例患者2年总生存（OS）率为28.8%（95%CI 13.5%~46.0%），2年无病生存（DFS）率为27.1%（95% CI 12.5%~44.0%）。18例仅使用化疗或化疗加靶向药物治疗的患者，17例在2年内死亡，1例放弃治疗后失访。接受allo-HSCT治疗的患者3年OS率为43.4%（95%CI 13.7%~70.4%），3年DFS率为42.7%（95% CI 13.4%~69.7%）。结论同时伴FLT3-ITD突变及MLL基因异常的AML患者FAB分型以M5多见，常伴高白细胞血症、细胞遗传学异常及多基因异常。患者化疗缓解率低，早期复发率高，长期生存率低。高白细胞血症、多基因异常可能是此类患者疗效差的重要原因，allo-HSCT可改善患者的转归。

3109 - BM-Mscs From Risk-Stratified AML Patients Support Normal Hematopoiesis and are Anti-Inflammatory in in Vivo Preclinical Models of Hematopoietic Reconstitution and Severe Colitis

Acute myeloid leukemia (AML) represents a heterogeneous group of disorders characterized by the rapid expansion of immature myeloid cells (blasts) in the bone marrow (BM). There is wide disease heterogeneity and patient risk-stratification principally relies on cytogenetic-molecular data. BM mesenchymal stem/stromal cells (BMSC) are key components of the hematopoietic niche thought to contribute to leukemia pathogenesis. We recently reported a correlation between behaviors of AML-derived BMSCs (AML-BMSCs) in vitro and overall survival in AML patients. To elaborate on this finding, we investigated the capacity of AML-BMSCs from risk-stratified de novo AML patients to regulate the homeostasis of healthy CD34+ hematopoietic stem/progenitor cells (HSPC), and to exert anti-inflammatory effects in vivo in a pre-clinical model of severe acute colitis. We report that regardless of risk-group, AML-BMSCs support, similar to HD-BMSCs, the survival, proliferation, differentiation and clonogenecity of CD34+ cells in vitro, and the in vivo immune deficient mice repopulating assays. Additionally, AML-BMSC were capable of reversing the inflammatory phenotype in preclinical models of acute severe colitis, showing a greater anti-inflammatory capacity compared to HD-BMSC. Collectively, these data indicate that AML-BMSCs retain the capacity to support healthy HPSC and can suppress inflammation in vivo.

Lymphocyte Count before First Consolidation Therapy Is A Predictor of Relapse free Survival in Acute Myeloid Leukemia

To investigate the effects of absolute lymphocyte count(ALC) before start of the first cycle of consolidation chemotherapy(CC) on the relapse free survival in the patients with acute myeloid leukemia(AML), so as to explore a simple and easy method for predicting AML relapse. The clinical data of 132 patients with newly diagnosed AML (all non-acute promyelotic leukemia) from 2011 to 2017 were analyzed retrospectively. The 132 AML patients were treated with standard induction chemotherapy (IC) and consolidation chemotherapy (CC). According to lymphocyte count of patients before start of the first cycle of CC, the AML patients were divided into 2 group: high lymphocyte count group (H-Lym≥1.2×109/L) and low lymphocyte count group (L-Lym<1.2×109/L). The differences in ralapse rate and relapse-free survival between 2 groups were analyzed. Among 132 patients with AML, patients who could be valuated and were elicible for the study accounted for 65 (49.24%). The absolute leukocyte count, age, chromosome karyotypes before IC of patients did not show statistical difference between H-Lym group (40 cases) and L-Lym group (25 cases). Unvarvate analysis showed that the Low lymphocyte count and unfavorable chromosome karyotypes were poor prognostic factors for the relapse-free survival time, and there was significant difference between 2 groups (P<0.01). The relapse risk in patients of L-Lym group increased, the hazard ratio (HR)=3.01 (95% CI=1.55-4.98) (P<0.01). In multivariate analysis containing unfavorable prognostic karyotypes, this trend still existed (HR=2.52, 95% CI 1.28-9.98)(P<0.01). The AML patients with high lymphocyte count before the first CC have more long relapse free survival time suggesting that the lymphocyte count before the first CC may be prognostic factor for relapse free survival of AML patients.

Correlation of TET2 SNP rs2454206 with improved survival in children with acute myeloid leukemia featuring intermediate-risk cytogenetics.

Single nucleotide polymorphisms (SNPs) may influence the disease course and outcome of hematologic neoplasms. SNP rs2454206 is common in the TET2 gene, which plays a role in epigenetic regulation of myelopoiesis. Few investigations examined the role of TET2 SNP rs2454206 in acute myeloid leukemia (AML) and none of those studies was performed in Chinese populations. Here, we report the prevalence and clinical relevance of TET2 SNP rs2454206 in 254 Chinese patients with childhood AML. Our data demonstrate that TET2 SNP rs2454206AG/GG is associated with improved overall survival and event-free survival in AML patients with intermediate-risk cytogenetics features. The prognostic impact of TET2 SNP rs2454206AG/GG was independent of other common AML risk factors, such as age, white blood cell count, and FLT3-ITD. No difference in TET2 expression levels in AML with TET2 SNP rs2454206AA and TET2 SNP rs2454206AG/GG was detected, indicating that TET2 SNP rs2454206 status does not affect TET2 expression in pediatric AML.

SPG6 supports development of acute myeloid leukemia by regulating BMPR2-Smad-Bcl-2/Bcl-xl signaling

Acute myeloid leukemia (AML) is the most common acute leukemia affecting adults. To effectively treat AML, new molecular targets and therapeutic approaches must be identified. In silico analysis of several available databases of AML patients showed that the expression of Spastic Paraplegia 6 Protein (SPG6) significantly inversely correlates with the overall survival of AML patients. To determine whether SPG6 supports AML development, we employed an shRNA-encoding lentivirus system to inhibit SPG6 expression in human AML cells including NB4 and MV4-11 cells. Knockdown expression of SPG6 resulted in decreased cell growth and elevated apoptosis of these leukemia cells. Notably, the SPG6 deficiency resulted in higher BMPR2 expression indicating that BMPR2 signaling contributes to AML pathogenesis. Furthermore, SPG6 deficiency promoted phosphorylation of Smad1/5/9 and decreased transcription of Bcl-2 and Bcl-xl. Our study suggests that SPG6 contributes to AML pathogenesis, and suggests that inhibition of SPG6 may be novel strategy for treating human AML.

BAALC and ERG expression levels at diagnosis have no prognosis impact on acute myeloid leukemia patients undergoing allogeneic hematopoietic stem cell transplantation.

Brain and acute leukemia, cytoplasmic (BAALC) and ETS-related gene (ERG) expression levels are independent prognostic factors for acute myeloid leukemia (AML); however, their prognostic impacts on AML patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) require further investigation. We studied 71 de novo AML patients treated with allo-HSCT and defined low and high expressers according to the median expression levels of BAALC and ERG at diagnosis respectively. High BAALC expression was associated with wild-type NPM1 (P = 0.000) and RUNX1 mutations (P = 0.027). High ERG expression was associated with FLT3-ITD absence (P = 0.003) and wild-type NPM1 (P = 0.001). BAALC and ERG expression levels were significantly correlated with each other (P = 0.001). Survival analyses including Kaplan-Meier curves and univariate and multivariate analysis consistently reported that there were no significant differences for both event-free survival (EFS) and overall survival (OS) (all P > 0.1), between high versus low BAALC and ERG expressers. Our study suggested that despite of their well-known adverse role in prognosis of AML, neither BAALC nor ERG expression levels at diagnosis had effect on survival of AML patients who underwent allo-HSCT.

A Promiscuous Biotin Ligase as a Global Strategy to Interrogate the Protein Interactome of Human ABCC4

ATP‐Binding Cassette transporter sub‐family C member 4, ABCC4, is a plasma membrane transporter with the ability to export prostaglandins and cancer chemotherapeutics. In acute myeloid leukemia (AML), ABCC4 interacts with a binding protein through its PDZ motif1. This interaction with a highly expressed PDZ protein, a protein which correlated with worse overall survival for AML patients, affects ABCC4 plasma membrane localization and retention, leading to profound effects on its function and drug resistance. The intense focus on ABCC4's PDZ motif as a locus for protein interaction has left other potential protein interactions that might regulate ABCC4 function largely unexplored. Little is actually known about how ABCC4 traffics to and from the plasma membrane. In the present study, we developed a global approach to not just identify potential interacting proteins, but also determine the proteins required for ABCC4 trafficking and internalization. We used a promiscuous biotin ligase, BioID2, which covalently attaches a biotin moiety to nearby proteins in the presence of biotin. Fusing BioID2 to the N‐terminus of ABCC4 did not alter its glycosylation, plasma membrane localization, or function. Upon addition of biotin, the BioID2‐ABCC4 fusion protein was capable of biotinylating itself as well as ABCC4 proximal and interacting proteins. We then developed an immunoprecipitation and elution protocol and strategy to specifically identify proteins biotinylated by the BioID2‐ABCC4 fusion protein. With successful immunoprecipitation of biotinylated proteins and identification by mass spectrometry, proteins within the vicinity of ABCC4, from its genesis at the endoplasmic reticulum to its final destination at the plasma membrane, will be identified. At the same time, proteins involved in ABCC4 recycling to degradation will also be discovered. Overall, this study will provide unique insights into the mechanisms of ABCC4 trafficking and identify proteins that will allow for modulation of ABCC4 level. This will be beneficial for malignancies associated with high ABCC4 expression, including neuroblastoma, prostate, non‐small cell lung, and endometrial cancers.Support or Funding InformationThis work was supported by NIH and ALSAC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.