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Introduction: Daratumumab is a human IgGκ monoclonal antibody targeting CD38 with a direct on-tumor and immunomodulatory mechanism of action. In phase 3 clinical studies, the addition of daratumumab to standard-of-care regimens consistently demonstrated a significant progression-free survival (PFS) benefit and improved depth of response, including minimal residual disease-negativity, in patients (pts) with newly diagnosed MM or RRMM. In the primary analysis of the phase 3 CASTOR study (median follow-up: 7.4 mo), D-Vd reduced the risk of disease progression or death by 61% (median PFS, not reached [NR] vs 7.2 mo; hazard ratio [HR], 0.39; 95% confidence interval [CI], 0.28-0.53; P Methods: Eligible pts in LEPUS and CASTOR received ≥1 prior line of therapy and were randomized 2:1 in LEPUS and 1:1 in CASTOR to 8, 21-day cycles of V (1.3 mg/m2 SC) on Days 1, 4, 8, and 11, and d (20 mg, PO or IV) on Days 1, 2, 4, 5, 8, 9, 11, and 12 ± D (16 mg/kg IV) given QW for Cycles 1-3, Q3W for Cycles 4-8, and Q4W thereafter. The primary endpoint for both studies was PFS. Results: A total of 211 (D-Vd, n=141; Vd, n=70) pts in LEPUS and 498 (D-Vd, n=251; Vd, n=247) pts in CASTOR were randomized. The median (range) age was 61 (28-82) years for Chinese and 64 (30-88) years for global pts. In general, baseline characteristics were similar between Chinese and global pts and balanced between treatment arms, with the exception of median body weight (LEPUS: 67 kg; CASTOR: 76 kg). Chinese and global pts both received a median of 2 prior lines of therapy; 79% and 66% received prior V, 27% and 28% were refractory to lenalidomide, and 64% and 32% were refractory to their last prior line of therapy, respectively. After a median follow up of 8.2 months in LEPUS and 7.4 months in CASTOR, a consistent PFS benefit of D-Vd vs Vd was demonstrated in the pooled analysis set across age (60 mL/min) subgroups (Table 1). Median time to progression was also consistently prolonged with D-Vd vs Vd across pooled pt subgroups. Among response evaluable pts, D-Vd improved overall response rate, rate of very good partial response or better, and rate of complete response or better (Table 2) and prolonged median duration of response vs Vd in all pt subgroups. Additional data, including PFS2, from the pooled subgroups analysis will be presented at the meeting. The safety profile of D-Vd was generally consistent across pts in LEPUS and CASTOR. Grade 3/4 treatment-emergent adverse events (TEAEs; D-Vd/Vd) occurring at a ≥5% frequency with D-Vd vs Vd in both Chinese and global pts included thrombocytopenia (51%/37%; 45%/33%), lymphopenia (44%/29%; 10%/3%), neutropenia (16%/6%; 13%/4%), and hypertension (12%/3%; 7%/1%). 49%/38% of Chinese and 42%/34% of global pts had ≥1 serious TEAEs. TEAEs leading to treatment discontinuation occurred in 4%/3% of Chinese and 7%/9% of global pts, and TEAEs leading to death occurred in 4%/10% of Chinese and 5%/6% of global pts. Rates of infusion-related reactions (IRRs) were similar for D-Vd across studies (LEPUS: 38%; CASTOR: 45%); most occurred during the first infusion and the majority were grade 1/2. Conclusions: D-Vd demonstrated a clinical benefit, including significantly improved PFS, in pooled Chinese and global pts with RRMM who received ≥1 prior line of therapy, regardless of age, cytogenetic risk status, or renal function. The safety profile of D-Vd was consistent across all pts, and no new safety concerns were identified. These results support the use of D-Vd in Chinese pts with RRMM. Disclosures Jin: The First Affiliated Hospital of Zhejiang University: Current Employment. Spencer:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Cilag GmbH: Consultancy, Honoraria, Research Funding, Speakers Bureau. Weisel:Roche: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Adaptive: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Honoraria. Mateos:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria; Regeneron: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Nooka:Adaptive Technologies: Consultancy, Honoraria; Spectrum Pharmaceuticals: Consultancy; Oncopeptides: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: Personal Fees: Travel/accomodations/expenses, Research Funding; Karyopharm Therapeutics, Adaptive technologies: Consultancy, Honoraria, Research Funding. Qi:Janssen: Current Employment, Current equity holder in publicly-traded company; Johnson and Johnson: Current equity holder in publicly-traded company. Sun:Janssen: Current Employment, Current equity holder in publicly-traded company. Gai:Janssen: Current Employment, Current equity holder in publicly-traded company. Liu:Janssen: Current Employment, Current equity holder in publicly-traded company. Yang:Janssen: Current Employment, Current equity holder in publicly-traded company.

High-risk B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive disease, often characterized by resistance to chemotherapy. A frequent feature of high-risk B-ALL is loss of function of the IKAROS (encoded by the IKZF1 gene) tumor suppressor. Here, we report that IKAROS regulates expression of the BCL2L1 gene (encodes the BCL-XL protein) in human B-ALL. Gain-of-function and loss-of-function experiments demonstrate that IKAROS binds to the BCL2L1 promoter, recruits histone deacetylase HDAC1, and represses BCL2L1 expression via chromatin remodeling. In leukemia, IKAROS’ function is impaired by oncogenic casein kinase II (CK2), which is overexpressed in B-ALL. Phosphorylation by CK2 reduces IKAROS binding and recruitment of HDAC1 to the BCL2L1 promoter. This results in a loss of IKAROS-mediated repression of BCL2L1 and increased expression of BCL-XL. Increased expression of BCL-XL and/or CK2, as well as reduced IKAROS expression, are associated with resistance to doxorubicin treatment. Molecular and pharmacological inhibition of CK2 with a specific inhibitor CX-4945, increases binding of IKAROS to the BCL2L1 promoter and enhances IKAROS-mediated repression of BCL2L1 in B-ALL. Treatment with CX-4945 increases sensitivity to doxorubicin in B-ALL, and reverses resistance to doxorubicin in multidrug-resistant B-ALL. Combination treatment with CX-4945 and doxorubicin show synergistic therapeutic effects in vitro and in preclinical models of high-risk B-ALL. Results reveal a novel signaling network that regulates chemoresistance in leukemia. These data lay the groundwork for clinical testing of a rationally designed, targeted therapy that combines the CK2 inhibitor, CX-4945, with doxorubicin for the treatment of hematopoietic malignancies.

Introduction T-cell acute lymphoblastic leukemia (T-ALL) is a common hematological malignancy with a high unfavorable prognosis. Ubiquitin-specific-processing protease 7 (USP7) is one of the deubiquitinating enzymes attracting concentrated attention in current studies for cancers; it is also involved in regulation of oncogenic transcriptional program in T-ALL, and serves as a potential target to treat T-ALL. USP7 is interacted with PHD Finger protein 8 (PHF8), a histone lysine demethylase in T-ALL. However, the impact of the interaction in T-ALL development is undetermined. In this study, we explored the anti-tumor effect of targeting USP7 and its downstream substrate in T-ALL and underlying mechanism. Methods In vitro GST pull-down assay, co-immunoprecipitation assay, protein deubiquitination and stability assays were used to detect the interaction of USP7 with its direct substrate and the ubiquitination level. Gain-of-function(overexpression) and loss-of-function (lentiviral shRNA knockdown) approaches for USP7 were conducted for gene expression, CCK-8 cell proliferation assay, and apoptosis assay with annexin V + PI staining. ChIP-qPCR assay was used to explore the enrichment of histone markers in promoter region of target gene. In vivo human T-ALL xenograft mouse model was developed in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse by tail vein injection of 2.5 × 10 6 /mouse and the leukemia engraftment was evaluated with human CD45+ cells by flow cytometry. The qPCR and western blot were carried out for detection mRNA level and protein level of related genes. The mRNA level of USP7 and its substrate PHF8 was examined by qPCR in 43 newly-diagnosed T-ALL patients with an approval of the Ethics Committee. Microarray datasets of T-ALL patients from GEO database were used to determine the USP7 and its substrate -related genes with web-based platform MEM and Limma. Results Results showed that USP7 directly interacted with PHF8; and overexpressed USP7 deubiquitinated and stabilized PHF8, and enhanced cell proliferation of JURKAT T-ALL cells. These data indicates targeting USP7 promotes cell proliferation of T-ALL cells through its substrate PHF8. Moreover, SNAI1 was identified as one of key co-expressed genes with PHF8 in microarray datasets from T-ALL patients, and knock-down of PHF8 suppressed the SNAL1 expression and increased the recruitment of repressive histone marker, H3K9me2 but decreased the enrichment of H3K4me3 in SNAI1 promoter. SNAI1 knockdown significantly induced the cell proliferation arrest and apoptosis of T-ALL cells (Fig.1A & 1B). With in vivo human leukemia xenograft mouse model, we further demonstrated that SNAI1 knockdown significantly reduce the spleen size and weight, the ratio of human CD45(+) cells, bone marrow cellularity and also the inflammatory cell infiltration compared to the scramble shRNA control (Fig.1C-1F). Moreover, Wnt signaling was identified as SNAI1 interaction partners by the pathway analysis in the SNAI1 interaction genes in the microarray data from T-ALL patients. SNAI1 knockdown significantly suppressed the expression of the Axin2 and Survivn, the key components of Wnt signaling. These data suggested oncogenic role of USP7/PHF8/SNAI1/Wnt signaling in T-ALL. Next, we explored the effect of SNAI1 on USP7 knockdown-induced anti-tumor effect. We found that USP7 knockdown induced cell proliferation arrest and apoptosis, and overexpression of SNAI1 could block the effect in vitro. Furthermore, the in vivo data showed that USP7 knockdown significantly suppressed spleen size and weight, the ratio of human CD45(+) cells, bone marrow cellularity, inflammatory cell infiltration, and the protein expression of PHF8, SNAI1, Axin2 and Survivin; and SNAI1 overexpression completely rescue the USP7 knockdown-mediated antitumor effect and restored the expression of Axin2 and Survivin in vivo (Fig.2). Conclusion: Our results demonstrated that targeting USP7 by shRNA induces the cell proliferation arrest and apoptosis by promoting ubiquitination of PHF8 and suppressing SNAI1/Wnt signaling in T-ALL. Our data also revealed the oncogenic roles of USP7/PHF8/SNAI1/Wnt signaling in T-ALL and suggested targeting the signaling pathway as potential therapy in T-ALL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2), which plays critical roles in transcription repressions.[1] PRC2 is required for acute Myeloid Leukemia (AML) cell survival, and EZH2 is overexpressed and related to worse prognosis in AML, therefore, EZH2 may involve in leukemogenesis through inhibition of the tumor suppressor genes in AML. [2, 3] BCL-2, one of the key anti-apoptosis proteins is dysregulated in AML. Venetoclax (Ven, ABT-199), a BCL-2 selective inhibitor has anti-tumor activity in AML but its overall response rate of monotherapy was unsatisfied owing to the clinical resistance to the inhibitor. [4] In this study, we examined the effect of targeting EZH2 on chemosensitivity of BCL-2 inhibitor in AML and the underlying mechanisms. Methods Cell Counting Kit-8 assay was used for cell proliferation and cytotoxicity in U937 and MV-4-11 AML cells treated with vehicle control, EZH2 inhibitor (DZNeP), Ven and Combination (Combo, Ven+DZNeP) for 48 hours. Synergistic effect was analyzed with Calcusyn. RNA-seq was performed with total RNA isolated from U937 cells treated with 2μM DZNeP, 7.5μM Ven or vehicle for 48 hours. Apoptosis was measured by cell staining with Annexin V+propidium iodide (PI) following flow cytometry analysis. EZH2 mRNA level was examined by qPCR in 39 newly-diagnosed AML patients from February 1, 2016 to February 28, 2019 at our institute with an approval of the Ethics Committee. Level of the apoptotic effectors was detected by western blot. GEPIA (Gene Expression Profiling Interactive Analysis) and R2 genomics analysis and visualization application were utilized for survival analysis. Results Both Ven and DZNeP had a dose-dependent and time-dependent effect on cell proliferation arrest in U937 and MV4-11 cells. DZNeP significantly sensitized the effect of Ven on cell proliferation arrest compared to single drug only (P Conclusions Our data demonstrate the DZNeP sensitizes the effect of Ven in AML. Our results also reveal a novel mechanism that accounts for the synergistic effect of the two drugs and the fact that DZNeP may increase the chemosensitivity of BCL-2 inhibitor through PIK3IP1-PI3K/AKT axis in AML. Our findings suggest the potential combined therapy of Ven+DZNeP for AML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: HMAs are mainstay treatment of higher-risk myelodysplastic syndromes (MDS). However, clinical outcomes of patients treated with decitabine (DEC) monotherapy were far from satisfactory with an overall response rate (ORR) of 33%-55.4% and an overall survival (OS) of 17.7-22 months. Some clinical researches reported that the addition of all-trans retinoic acid (ATRA) to DEC increased response rate and prolonged survival of MDS and elderly acute myeloid leukemia (AML) patients. Our data showed ATRA enhanced the cytotoxic effect of DEC on MDS via activating RARα-Nrf2 complex (2021 EHA abstract EP891). These findings suggested that addition of ATRA to DEC in treatment-naive patients may improve response rate based on the synergetic function. We therefore conducted a study of combination of DEC and ATRA in MDS subtype excessive blasts (EB) patients. Methods: In this randomized, multicenter, open-label trial, patients with newly diagnosed MDS subtype EB based on the 2016 WHO classification from 7 different tertiary medical centers in China were included. Patients were randomized 1:1 to receive either oral ATRA (25mg/m 2/day on days 1-28) plus DEC (20 mg/m 2 daily on days 1-5) or DEC monotherapy（Figure 1）. The primary endpoint was overall response rate ORR, defined as complete remission (CR), partial remission (PR), marrow complete remission (mCR), or hematological improvement (HI). Key secondary endpoints were mCR, HI, overall survival (OS), and progress free survival (PFS). Response was assessed after completion of four cycles of treatment. For patients who bridged to allo-HSCT later on and did not complete four cycles of treatments, response was defined as best response ever observed before receiving allo-HSCT. Here, we report results of the interim analysis. Results: Between May 2018 and July 2021, 165 patients were randomly allocated into either DEC plus ATRA (n=82) or DEC monotherapy (n=83). 63.6% of patients were male and 36.4% were female, with a median age of 62 years (range, 19 to 81 years). 38.8% of patients had EB1 and 61.2% had EB2. As of July 31, 2021, 126 patients were available for the assessment of treatment results. After a median follow up of 9.6 months, median number of courses on treatment was 4 courses (range, 1-14), 61 in DEC plus ATRA arm and 65 in DEC arm . OR was achieved in 85.2% of DEC plus ATRA patients compared to 56.9% in DEC monotherapy (p 72.7% patients developed at least one adverse event (AE) during the trial, 73.3% in the DEC plus ATRA arm and 72.0% in DEC arm, respectively. Grade 3/4 Hematological toxicity occurred in 73.68 % of DEC plus ATRA arm and 76.92 % of DEC arm. Hyperlipidemia occurred in 31 patients in DEC plus ATRA arm (3.2% grade 3/4), and 18 (no grade 3/4) in DEC arm (p Conclusion: Combination of DEC and ATRA achieved an OR rate of 85.2% in MDS subtype EB. Enrollment is ongoing to assess its efficacy and safety in treating EB. This therapy may be a new treatment option for EB subjects. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: Severe aplastic anemia (SAA) is a hematologic disorder characterized by bone marrow hypoplasia and peripheral pancytopenia. Immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and cyclosporine (CsA) is the standard first-line treatment for patients with SAA who are ineligible for hematopoietic stem cell transplantation (HSCT). For those refractory to or relapsed after IST, eltrombopag (an oral thrombopoietin receptor agonist) were recommended as the second-line therapy by US FDA and European Medicines Agency (EMA). However, in several countries including China, eltrombopag has not been approved for use in patients with SAA, so there is an urgent unmet medical need for additional efficient treatment options. Hetrombopag, another oral thrombopoietin receptor agonist, has similar mechanism in stimulating thrombopoietin receptor signaling pathway as eltrombopag and better pharmacological performance than eltrombopag in pre-clinical study (Xie et al, JCMM 2018). In a phase 1 trial, hetrombopag was safe and well-tolerated in healthy subjects with potent thrombopoietic activity and acceptable pharmacokinetic profiles (Zheng et al, BCPT 2017). Here, in this phase 2 trial (NCT03557099), we aimed to assess the activity and safety of hetrombopag in patients with SAA who have had an insufficient response to IST. Methods: This open-label, single-arm, phase 2 study enrolled patients with SAA who have had an insufficient response to IST and were ineligible for transplantation. Patients were given hetrombopag orally at an initial dose of 7.5 mg once daily after overnight fasting. If increase of platelet count was less than 20×109/L from baseline, the dose of hetrombopag was gradually up-titrated by 2.5 mg every 2 weeks to a maximum of 15 mg once daily, for a total of 52 weeks. The primary endpoint was proportion of patients achieving hematologic responses in at least one lineage at week 18. Results: Between June 20, 2018 and May 24, 2019, 55 eligible patients were enrolled and received hetrombopag treatment. The median age was 40 years (range 19-65) and 34 (61.8%) patients were male. As of data cutoff on June 12, 2020, 52 (94.5%) patients received the maximum dose of 15 mg once daily, and the final dose of the other three patients was 7.5 mg, 10 mg, and 12.5 mg, respectively. A total of 24 (43.6%, 95% CI 30.3-57.7) of the 55 patients met primary endpoint, achieving at least one lineage hematologic response at week 18 after the initiation of hetrombopag treatment (Figure 1). Of them, 11 (20.0%) patients had unilineage responses, 7 (12.7%) had bilineage responses, and 6 (10.9%) had trilineage responses. Twenty-four (43.6%, 95% CI 30.3-57.7) patients responded in at least one lineage at week 24 and 27 (49.1%, 95% CI 35.4-62.9) patients responded in at least one lineage at week 52 (Figure 1). Of the 32 patients with responses at week 18, 24, or 52, median time to initial hematologic response was 7.9 weeks (range 2.0-32.1). The responses were durable in majority of patients who remained on hetrombopag treatment, with only six patients relapsed, achieving a probability of recurrence-free survival at 12-month of 79.5% (95% CI 59.9-90.3). Adverse events of any attribution were reported in 54 (98.2%) of the 55 patients, with the most common ones being upper respiratory tract infection (22, 40.0%), increased aspartate aminotransferase (12, 21.8%), and pyrexia (12, 21.8%). Twenty-eight (50.9%) patients had treatment-related adverse events. Adverse events of grade 3 or higher occurred in 17 (30.9%) patients, with only one (1.8%) related to hetrombopag. Three (5.5%) patients underwent dose reduction or treatment interruption and three (5.5%) patients were discontinued from treatment because of adverse events, but none of which was considered as drug-related. Fifteen (27.3%) patients had serious adverse events. Three (5.5%) deaths were reported, and all of them were judged to be not treatment-related. Clonal cytogenetic evolution occurred in two (3.6%) patients during hetrombopag administration, with one patient had a loss of chromosome 7 and one had an increase of chromosome 8. Conclusions: Hetrombopag showed encouraging activity with multilineage hematologic responses in patients with SAA who have had an insufficient response to prior IST. Hetrombopag was well-tolerated and safe. This study provided evidences that hetrombopag might represent a novel treatment option for patients with SAA. Disclosures He: LongBio Pharma: Consultancy, Research Funding; F. Hoffmann-La Roche: Consultancy, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland. Shen:Jiangsu Hengrui Medicine Co., Ltd: Current Employment. Tai:Jiangsu Hengrui Medicine Co., Ltd: Current Employment. Zhang:Jiangsu Hengrui Medicine Co., Ltd: Current Employment. Zhu:Jiangsu Hengrui Medicine Co., Ltd: Current Employment. Zou:Jiangsu Hengrui Medicine Co., Ltd: Current Employment.

Ikaros (IKZF1) is a tumor suppressor that binds DNA and regulates expression of its target genes. The mechanism of Ikaros activity as a tumor suppressor and the regulation of Ikaros function in leukemia are unknown. Here, we demonstrate that Ikaros controls cellular proliferation by repressing expression of genes that promote cell cycle progression and the phosphatidylinositol-3 kinase (PI3K) pathway. We show that Ikaros function is impaired by the pro-oncogenic casein kinase II (CK2), and that CK2 is overexpressed in leukemia. CK2 inhibition restores Ikaros function as transcriptional repressor of cell cycle and PI3K pathway genes, resulting in an antileukemia effect. In high-risk leukemia where one IKZF1 allele has been deleted, CK2 inhibition restores the transcriptional repressor function of the remaining wild-type IKZF1 allele. CK2 inhibition demonstrated a potent therapeutic effect in a panel of patient-derived primary high-risk B-cell acute lymphoblastic leukemia xenografts as indicated by prolonged survival and a reduction of leukemia burden. We demonstrate the efficacy of a novel therapeutic approach for high-risk leukemia: restoration of Ikaros tumor suppressor activity via inhibition of CK2. These results provide a rationale for the use of CK2 inhibitors in clinical trials for high-risk leukemia, including cases with deletion of one IKZF1 allele.

Background: The relapse rate is around 20-40% in adult B-cell acute lymphoblastic leukemia (B-ALL). The genetic defects are the major reasons for the relapse and poor outcome. We screened the genomic variants with Pan-cancer panel in B-ALL patients and whole exome-seq (WES) in the paired de novo-relapsed B-ALL samples. Methods: The xGen Pan-Cancer Panel (IDT), which has been designed with the probes targeting 127 significantly mutated genes from the TCGA database across 12 tumor tissue types (Nature, 502:333-339) was used. Agilent SureSelect Human All Exon V4+UTRs (Agilent) was used to target coding exons plus UTRs for the WES analysis. The genomic DNA from 81 Philadelphia chromosome positive (Ph+) B-ALL patient samples (71 de novo and 10 relapsed, 14 to 77 years old) collected between June 2008 and June 2016 at Zhongda Hospital Southeast University were used for the Pan-cancer panel screening. All DNA samples were sheared and generated approximately 260bp DNA fragments. The fragmented DNA was processed into Illumina compatible sequencing libraries using the Kapa Hyper Prep Kit. Each library was uniquely barcoded and captured by either the Pan-cancer panel or WES probes, followed by PCR amplification and sequencing on a HiSeq 2500 (Illumina) with 2x100 bp reads. The sequencing reads were aligned to the human genome (hg19) by following Broad Institute's GATK best practice pipeline to call germline short variants (SNPs and Indels). Called variants were annotated using ANNOVAR (version 2.3). Variants with exonic, nonsynonymous, stopgain, or stoploss, novel SNPs (not covered by dbSNP138), and with predicted deleterious/damaging functions were manually surveyed by IGV to confirm. Two paired de novo-relapse samples from Ph(+) B-ALL patients were performed the WES analysis. Results: We identified a total of 3945 single nucleotide variants (SNVs), 2222 insertions and deletions (INDELs) in the Pan-cancel panel analysis in 81 Ph(+) B-ALL patients. Among these, 101 genomic variants are with amino acid change, 8 are with stopgain, and 5944 have not been previously reported. We evaluated the frequency and distribution of likely pathogenic variants (PVs) detected in the cohort. Likely PVs were defined by SIFT algorithm which predicts whether an amino acid substitution is likely to affect protein function. Defined by the SIFT's qualitative score 'deleterious', we detected 46 PVs. Among these, PVs were commonly detected in KMT2C, APC, CDKN1A, NSD, BRCA1, EPHA3, and PIK3CG. The PVs were also validated with the Sanger DNA sequencing in the patients. The patients with the likely PVs have significantly higher WBC count (61*10^9/L vs. 24.45*10^9/L, P=0.004). Survival analysis showed that the patients with likely PVs had a worse event-free survival (EFS) and overall survival (OS), the difference was statistically significant (8 months vs. 15 months, P=0.017 and 14 months vs. 25 months, P=0.021). In order to gain an insight to the gene mutations contributing to the disease relapse, we compared the mutants spectrum between the de novo and relapsed samples. We found the genomic variants in NF1, CDK12, mTOR, and USP9X genes appeared mostly in relapsed samples, indicating their roles in the relapse. Using the WES, we further analyzed the genomic variants in two paired de novo and relapsed samples. We detected totally 40354 (de novo) and 16822 (relapsed) genomic variants, and among these 10415 (de novo) and 3082 (relapsed) are with amino acid change in patient 1; likewise, 30130 (de novo) and 14003 (relapse), and among these 7200 (de novo) 2534 (relapsed) with amino acid changes in patient 2. Totally 216 genomic variants with amino acid changes in 162 genes appeared only in the two relapsed samples, among which 10 genomic variants in ADAMTS8, CDK11B, EFCAB4B, FBXL21, HYDIN, IRF2BPL, MIR205HG, PRIM2, ZNF717, ZNF880 appeared in both relapsed samples, revealing their driver roles in relapse. Also, 110 of the 216 genomic variants are not previously reported. Conclusion: Genomic variants in common human cancer driver genes were also detected in B-ALL patients. The new genomic variants detected in the relapse samples may be involved in the oncogenesis of the relapse, which will be further verified with functional analysis. Our data also suggested the significance of developing more efficient new therapies to prevent the relapse in hematological malignancies. Disclosures No relevant conflicts of interest to declare.

Backgroud: Cytokine receptor-like factor 2 (CRLF2) plays an important role in the development of normal B lymphocytes, which can mediate the proliferation of early B cells. However, the diect oncogenic effect of CRLF2 overexpression in acute lymhpoblastic leukemia (ALL) is far yet to be clarified. Here, we explored the effect of CRLF2 overexpression on cell proliferation and the effect of the novel JAK2 inhibitor on B-ALL cells with CRLF2 overexpression. Methods: The 83 patients with newly-diagnosed ALL (56 B-cell and 27 T-cell ALL; range from 14 to 77 years old) between June 2008 and June 2016 were studied at Zhongda Hospital Southeast University. The 21 normal bone marrow subjects were enrolled as controls. The qPCR method is developed for detection CRLF2 expression and the CRLF2 overexpression was determined with a cutoff value more than the highest sample of normal bone marrow control. Median differences between the cohorts were evaluated using a Mann-Whitney U-test. Frequency differences were analyzed using uni- and multivariate Cox model. Event-free survival (EFS) and overall survival (OS) were estimated by the Kaplan-Meier method and compared by log-rank test. CRLF2 F232C gain-of-function mutant which we previously reported or CRLF2 were expressed in Nalm6 and 697 B-ALL cells with lentiviral transduction. WST-1 cell proliferation assay and in vitro clonogenic assay were performed upon JAK2 inhibitor (BBT594) treatment. Nalm6-CRLF2-luc, Nalm6-F232C-luc, and Nalm6-vector-luc cells were injected via tail vein into the NSG mice. The leukemia engraftment was monitored once a week by living imaging. Results: The expression of CRLF2 in patietns with ALL was significantly higher than the normal control (P Conclusion: We showed that CRLF2 overexpression could enhance the proliferation and infiltration of human B-ALL cells, and for the first time indicated that JAK2 inhibitor could suppress the cell proliferation and clonogenesis of the CRLF2 overexpressed B-ALL cells. Our data provide direct evidence of the oncogenic role of CRLF2 overexpression and the new therapeutic potential for targeting CRLF2 overexpressed B-ALL with JAK2 inhibitor. Disclosures No relevant conflicts of interest to declare.

Background:T-cell malignancies are characterized by the excessive proliferation of hematopoietic precursor cells of T-cell lineage lymphocytes in bone marrow. T-cell malignancies consist of T-cell lymphoblastic leukemia (T-ALL) and T-cell non-Hodgkin lymphoma (T-NHL). Effective therapeutic options are limited for T-cell malignancies patients. Apoptosis is one of the most important means to cure cancer. Autophagy, a conserved process that sequesters and targets cellular components for lysosomal degradation, also plays highly context-specific roles in mediating cell death. Our research found that LW-218, a new synthetic flavonoid compound, exerted anti-T-cell malignancies cell lines effects by autophagy-mediated apoptosis induction. Methods: In vitrostudy, T-ALL cell lines (Jurkat, Molt4) and T-NHL cell lines (Hut102, Hut78) were used to investigated LW-218-induced apoptosis effects by survival assays (Flow cytometry, Annexin V/PI). A series of experiment were used to assess the autophagy flux triggered by LW-218, by using mCherry-GFP-LC3 construct, Lysotracker Red staining, and determining the expression ratio of LC3-II/LC3-I. Western blot assay, RT-qPCR, transfection with siRNA, and immunofluorescence were used to investigate the underlying mechanism. In vivostudies, the anti-tumor effects of LW-218 were evaluate in T-ALL xenografts which established by subcutaneous injection of Jurkat cells. Results:Mechanistic studies demonstrated that LW-218-induced cell apoptosis in T-cell malignancies cell lines was correlated with activation of caspase 8, caspase 3 and caspases 9, and the substrate PARP1. In addition, LW-218 induced autophagy flux in T-cell malignancies, as increased ratios of LC3B-II/LC3B-I and reductions of P62 expression. The increased numbers of GFP-mCherry puncta and fluorescent intensity also indicated the increase in autolysosomes. The autophagy-mediated cell death must strictly follow the criteria that the inhibition of autophagy, through either genetic or chemical means, prevents cell death. LW-218-induced cell apoptosis and the activation of caspases3/8/9 could be prevented when the autophagy flux was blocked by autophagy inhibitors. mTOR is a master regulator of cellular metabolism and governs the programmed cell death pathways of apoptosis and autophagy. The results showed that LW-218 inhibited the expression of p-mTOR (Ser2448). And the restored activation of mTOR correlated with the inhibition of autophagy flux, which indicated mTOR might be involved in LW-218-induced autophagy. LW-218 also induced cleavage of Bid, the substrate of caspases 8, which indicated that the apoptosis mediated by autophagy might via caspases 8 and Bid activation. Thein vivostudies also verified that LW-218 inhibited the growth of tumors. Conclusions:Our study provides a new insight into the mechanism of LW-218-induced autophagy-mediated apoptosis, suggesting the potency of LW-218 to be a promising agent against T-cell malignancies. Disclosures No relevant conflicts of interest to declare.

Corresponding author: Baoan Chen, MD, PhD; E-mail: cba8888@hotmail.com Zhuyuan Wang, PhD; E-mail: wangzy@seu.edu.cn Yiping Cui, PhD; E-mail: cyp@seu.edu.cn Keywords: leukemia; multi-drug resistance; P-glycoprotein; surface-enhanced Raman scattering; SERS intensity. Background Acquisition of multidrug resistance (MDR) in the chemotherapy of leukemia could decrease the survival rate of refractory/relapsing patients. One of the best characterized mechanisms of MDR in leukemia is mediated by multidrug resistance protein-1 and its product, P-glycoprotein (P-gp). Thus, accurate detection of P-gp is necessary for MDR diagnosis. In the recent years, surface-enhanced Raman scattering (SERS) has emerged as a new detection technology of biological label for immunoassay with the advantages of ultrasensitive screening ability and extensive adaptability. However, few researches have focused on the application of SERS immunoassay in the diagnostics of leukemia MDR. The aim of our study is to investigate the expressions of P-gp on the cell surface of K562/ADM cells, and in the whole-blood samples of leukemia using a SERS-based immunoassay technique. Methods To simulate the MDR occurrence, we mixed the K562 and K562/ADM cells at different ratios. Besides, we built up the concentration gradient of K562/ADM cells for the quantitative analysis. We also divided 30 blood samples (AML n=14, ALL n=16; female n=12, male n=18; age Results There were positive and stable SERS signals of peak intensity at 1078 cm-1 after suspended with target samples. First, the SERS intensity of K562/ADM was significantly higher than that of K562 (P Conclusion We have proposed a SERS-based immunoassay to evaluate the expression of P-gp, a product of MDR protein of leukemia. Qualitative and quantitative analysis of K562/ADM indicated excellent specificity, high sensitivity and detection limit, as well as great reproducibility of this immunoassay. It was also demonstrated that this immunoassay was with acceptable accuracy and detection reproducibility for clinical whole blood samples, which was of great importance and convenience for practical clinical application. These features have made SERS-based immunoassay a selective and convenient technique for the identification of leukemia MDR diagnosis. Disclosures No relevant conflicts of interest to declare.

Background: SH2B3 (Src homology 2 B3), an adaptor protein can suppress activation of the JAK-STAT signaling pathway and the cytokine signaling pathway. Co-existence of SH2B3 mutations with IKZF1 deletion and JAK mutations are involved in oncogenesis in myeloproliferative neoplasms (MPN) and acute lymphoblastic leukemia (ALL).SH2B3 mutations and their clinical significance in adult ALL patients are still not well understood. Methods: 106 patients with newly-diagnosed ALL (79 B-cell and 27 T-cell ALL; 14-77 years old) between June 2008 and June 2016 were studied at Zhongda Hospital Southeast University. The 15 normal bone marrow subjects were enrolled as controls. The study was approved by the Ethics Committee of the institute. The 1-7 exons of SH2B3 were amplified from the genomic DNA extracted from fresh mononuclear cells of the patients by polymerase chain reaction (PCR) and nested PCR. The PCR products were extracted by gel purification and sequenced. Median differences between the cohorts were evaluated using a Mann-Whitney U-test. Frequency differences were analyzed using uni- and multivariate Cox model. Relapse-free survival (RFS) and overall survival (OS) were estimated by the Kaplan-Meier method and compared by log-rank test. Results: Two kinds of SH2B3 mutations (c.724C>T, p.P242S; c.724C>T, p.P242S ) were detected in 107 ALL patients. The two SH2B3 SNPs were detected in 20 patients with B-ALL, which represents a detection rate is 25.6% (20/79), and in 4 patients with T-ALL, equating to a detection rate of 14.8% (4/27). There was no statistical difference between the detection rate in B-ALL and T-ALL (P>0.05). Both of the SH2B3 SNPs localize in the PH domain. The c.724C>T, p.P242S (rs78894077) on exon2 (n=23, 18.69%), are only detected in the heterozygous genotype (CT). The % CD13 (+) was lower in patients with the SH2B3 P242S mutation than those without the mutation (20% vs. 47%, P=0.031). Deletion of IKZF1, the Ikaros gene is associated with poor prognosis. IK6, is one of the most common and important types of the gene deletion. In our study, IK6 was detected in 13 of 72 ALL samples with adequate DNA. In these 13 patients, 4 of them co-existed with a SH2B3 P242S mutation. No significant difference of prognosis was observed in the 4 patients compared to those lacking SH2B3P242S mutation. However, in the 59 patients without IK6, there were 13 patients harboring the SH2B3 P242S mutant. The EFS (18 months vs. 5 months, P=0.019, OR=0.471, 95%CI= [0.233-0.950]) and OS (32 months vs. 13 months, P=0.038, OR=0.382, 95%CI= [0.146-1.003]) in these patients were significantly longer than the other 46 patients without a SH2B3 mutation. This data indicates that the SH2B3 P242S mutation may be associated with a better prognosis in patients. The c.784C>T, p.R262W on exon4 is detected in 1 Chinese patient (0.93%). There is also a rs3184504 (c.784T>C) SNP recorded in the database. However, we detected both homozygous (CC) and heterozygous (CT) genotypes on the nucleotide 784 site in this cohort. The CC genotypes was observed in 100% (15/15) of the normal controls and 99.07% of the ALL patients. Only one patient (0.93%) harbored the CT genotype. This data indicates that in the Chinese Han population, the CC genotype is dominant, and it should be the wild type genotype. The wild type for the Chinese Han population for the SH2B3 amino acid residue 262 is arginine "R" not tryptophan "W". The "W" in this site is the SH2B3 SNP (R262W) in the population. The patient with this SNP was a 62 year old male. He had a high WBC count (123*10^9/L), a low PLT count (25*10^9/L), BCR-ABL1 fusion (FISH: BCR-ABL1: 220/300), and also had expression of myeloid markers (CD13: 53.9%)when diagnosed. In addition, this patient had co-existing IL7R mutations (c.197T>C, p.I66T; c.254G>A, p.V138I and c.337C>T, p.H165H ) and an IKZF1 deletion, both of which are important members of the JAK-STAT signaling pathway. The patient relapsed 3 times within one year, and died with central nervous system leukemia. Certainly a bigger ALL cohort is needed to confirm if SH2B3 R262W SNP is related to relapse and poor prognosis. Conclusion: Two novel SH2B3 mutations were identified in a Chinese adult ALL cohort study. Our data showed that the different SH2B3 mutations have distinct clinical features, and the SH2B3 mutations may be used as new molecular markers for clinical risk stratification and even prognosis evaluation following a much bigger cohort validation. Disclosures No relevant conflicts of interest to declare.

Abstract Text: Objective: The mortality rate of refractory/relapsed acute myeloid leukemia (AML) is very high with poor prognostic. Three are still no standard treatments for these patients nowadays. Our research compared the treatment effect between patients with chemotherapy and transplantation. Method: A retrospective research of 45 patients with refractory/relapsed AML was performed in our center. We analyzed the clinical features including gender, age, classification of AML, performance status (PS), complete remission (CR) duration, cytogenetic and molecular abnormities. Survival analyses were made by using the Kaplan Meier method and took the Log - rank test. Results: The mean survival time of the 45 patients with refractory/relapsed AML was (36.25¡À8.40) months and the median follow up was (9¡À2.58) months. The one year and two years overall survival rate was (40.6¡À7.5)% and (23.7¡À7.0)%. Univariate analysis results demonstrated that age¡Ý60 years and failing to undergo HSCT after relapse had poor influence on OS. Conclusion: Performance of HSCT after relapse can improve the prognosis through the single center study. HSCT is still the effective salvage therapy for patients with refractory/relapsed AML. Our research is benefit for individual therapy and can be applied to improve the survival. Disclosures No relevant conflicts of interest to declare.

Multiple myeloma (MM) is a plasma cell malignancy that accounts for approximately 10% of all hematological cancer. Although over the last few decades significant improvement in outcomes has been observed in MM patients, the prognosis of MM remains unfavorable. Existing agents, including the proteasome inhibitor bortezomib and the immunomodulatory agents thalidomide and lenalidomide, have improved outcomes in patients with RRMM greatly. However MM still remains incurable and continuing treatment escalates in complexity while presenting a special therapeutic challenge for patients who these agents have failed. Carfilzomib, a proteasome inhibitor, was approval in 2012 in the United States for RRMM based on efficacy results. Recently, carfilzomib has become a promising therapeutic approach for relapsed and (or) refractory multiple myeloma (RRMM), but no study has summarized the overall effect of carfilzomib in RRMM. To explore the role of carfilzomib, we performed a meta analysis of all known prospective clinical trials to assess the efficacy of carfilzomib in patients with RRMM. Methods and Materials: A systematic review of publications in the PubMed, Embase, the Cochrane Library and ISI Web of knowledge was performed on September 15, 2015 according to the Preferred Reporting Items for Systematic Reviews and Meta Analysis (PRISMA) guidelines. Accounting for some of the inter-study variation, the random-effects model was chosen for the entire study to increase power and precision regardless of heterogeneity. All statistical analyses were conducted by using the STATA software. Meta analyses were carried out to calculate the overall response rate (ORR), complete response rate (CRR) and clinical benefit rate (CBR) of carfilzomib for RRMM. Results: Seven single-arm pilot studies and one randomized controlled trial (RCT) were included. Eight prospective studies enrolled a total of 1,446 patients with 1,000 evaluable patients. The overall quality of the seven single-arm pilot studies was moderate according to Newcastle-Ottawa scale. In the only randomized controlled trial including 792 patients, 396 patients were treated by carfilzomib with lethalidomide and dexamethasome. The quality of this study was adequate according to Cochrane tool for assessment of bias. In patients with RRMM, ORR was 0.44, CRR was 0.13 and CBR was 0.54. High heterogeneity between studies was observed, and funnel plots was symmetrical, negating publication bias. he safety of carfizomib was deemed good and no long-term complications were reported. In the eight prospective studies selected for this analysis, common adverse effect (AE) of the patients varied in different studies, including fatigue, nausea, anemia, thrombocytopenia, neutropenia, diarrhea, etc. Conclusion: In this comprehensive meta analysis, we evaluated the efficacy of carfizomib in the treatment of RRMM. Our meta analysis of the eight studies included, our results demonstrate that carfilzomib is a safe, effective and well tolerated treatment in a large, well-characterized group of patients with RRMM. The lack of severe toxicities observed in patients treated with carfilzomib indicates the potential for full doses of carfilzomib to be used for patients with advanced MM. Disclosures No relevant conflicts of interest to declare.

Information A simple, novel and fast electrochemical method was applied for clinical diagnosis of leukemia. Blood samples of leukemia patients were identified by screen-printed electrodes with potassium ferricyanide (III) probes. Our study successfully developed and raised the possibility of utilizing the electrodes in the identification and diagnosis of leukemia. Background Accurate preliminary detection of leukemia is vital for timely and suitable chemotherapy and clinical outcomes of patients. Electrochemical methods have been widely used in cytobiology, including identification of leukemia cells. In the present study, we creatively applied the use of an electrochemical method to the analysis of human blood samples. The aim of this study was to identify leukemia, breast cancer, respiratory infection and healthy individuals by detecting the electrochemical characteristics of leukocytes in the peripheral blood of patients with a screen-printed electrode. This method might be effectively used in the preliminary screening of leukemia. Methods A total of 99 blood samples obtained from Zhongda hospital in China (May 2016 - July 2016) were divided into four groups: healthy individuals (n = 25), respiratory infection (n=23), leukemia (n = 29) and breast cancer (n = 22). We used probes to distinguish leukocytes of people's blood from different samples immediately due to the different electrochemical behaviors of leukocytes. A screen-printed electrode was used to measure differential pulse voltammetry by a potassium ferricyanide (III) probe combined with a simple bio-sensor system. Then, one-way analysis of variance (ANOVA) method was applied to analyze the scanning curves and peak potential and peak shifts were compared (ΔEp). Results The scanning curves demonstrated the specific electrochemical behaviors of the blank potassium ferricyanide solution and which mixed with different samples in different groups. Significant differences in mean peak potentials were observed over the four groups (P< = 0.001). 105.00±8.02mV, 111.84±9.53mV, 120.90±11.18mV, 132.84±11.53mV for Group healthy, Group respiratory infection, Group leukemia, and Group breast cancer, respectively. Meanwhile, there were 4.67±3.14mV, 10.91±5.81mV,20.42±8.50mV, 33.42±10.05mV in peak shifts for above four groups respectively. Conclusions Using the screen-printed electrode to identify cancer was a novel method with good sensitivity and specificity. It might be effective and had a potential utility in the preliminary screening of leukemia. Disclosures No relevant conflicts of interest to declare.

Background: Allogeneic stem cell transplantation (allo-HSCT) is the only curative option for several hematological malignancies. Allogeneic stem cell transplantation (allo-HSCT) is the only curative option for several hematological malignancies. Patients with GVHD suffer from considerable organ damage, leading to significant morbidity, frequent hospitalizations and high mortality rates. Unlike the acute GVHD which is mediated by cytotoxic T-cell attack on host tissues, pathophysiology of cGVHD is significantly more complex and the mechanisms of dysregulated adaptive and innate immune responses are poorly understood. Many patients require additional salvage therapy for corticosteroid-refractory GVHD and have a dismal prognosis. In recent years, ECP as a therapeutic option has been widely studied in patients with GVHD. In particular, ECP has demonstrated positive effects in patients with steroid-refractory or steroid-intolerant chronic GVHD. The mechanism of action in ECP still remains to be elucidated. To investigate the evidence for clinical efficacy of ECP in the treatment of steroid-refractory cGVHD, we conducted a systematic search and meta-analysis comprising several databases and abstracts presented at recent annual meetings in the field. Methods and Materials: A systematic review of publications indexed in the PubMed, Embase, the Cochrane-controlled trails registry, the Cochrane Library and ISI Web of knowledge were performed on January 10, 2015 according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) guidelines. The use of a random-effects model should account for some of the inter-study variation, and all statistical analyses were conducted by using the STATA software. We estimated relative risk (RR) with their 95% confidence interval (95% CI) using the standardized mean difference (SMD). Heterogeneity was evaluated by using I2 values. Meta-analyses were carried out to calculate the overall response rate (ORR) and complete response rate (CRR) of cGVHD and the organ specific responses. Results: Eight complete peer-reviewed papers including seven single-arm pilot studies and one randomized controlled trial (RCT) met the selection criteria and were selected in this meta-analysis. In patients with cGVHD, pooled ORR and CRRwere 0.66 and 0.46 respectively, with organ specific responses of 0.80 for skin, 0.79 for gut, 0.57 for oral mucosa, 0.57 for liver, 0.50 for eye and 0.46 for joint involvement. The safety of ECP is excellent. No long-term complications were reported when compared to other immunosuppressive therapies. Conclusion: Compared with other meta-analyses, our meta-analysis included the most comprehensive literature in the field of ECP treatment in steroid-refractory cGVHD, collected the most recent papers, thus assessing the largest cohort ever of ECP patients (n=176), and employed modern statistical methods like funnel plots to evaluate heterogeneity of studies on the effect of ECP in patients with cGVHD thoroughly in our analysis, which could better help guide clinicians in the ECP application. Our analysis revealed a promising clinical benefit of ECP for patients with steroid-refractory cGVHD. Further prospective trials with larger cohorts are mandatory. Disclosures No relevant conflicts of interest to declare.

Background: Dynamin-2 (DNM2) is a GTPase essential for intracellular vesicle formation and trafficking, cytokinesis and receptor endocytosis. Mutations in DNM2 are common in early T-cell precursor acute lymphoblastic leukemia (ALL). However, DNM2 expression in other types of ALL is not reported. Ikaros, encoded by IKZF1, is a transcriptor factor functioned as a tumor suppress gene, and its dysfunction is associated with poor survival and high relapse rate in ALL. Casein Kinase II (CK2) inhibition could restore Ikaros function in high-risk leukemia and CK2 inhibitor-CX4945 showed the therapeutic efficacy on high-risk leukemia with human-derived xenograft mouse model. It is still undetermined if Ikaros regulates DNM2 expression in the leukemic cells. Methods: The 151 patients' and 30 volunteers' BM samples were collected between June 2008 and June 2014 at the First Affiliated Hospital of Nanjing Medical University. The ALL diagnosis was made according to the morphologic, Immunophenotypic, cytogenetic, and molecular criteria of WHO Diagnosis and Classification of ALL (2008).Cytogenetic and molecular analyses as previously reported. The DNM2 expression was determined by qPCR in the patients. All the patients were divided into high or low DNM2 expression groups (Q4 vs Q1-3) and the cutoff was determined by SPSS 17.0. For quantitative parameters, overall differences between the cohorts were evaluated using a Mann - Whitney U -test. For qualitative parameters, overall group differences were analyzed using a χ2 test. All statistical analyses were performed using the SPSS 17.0 and P Results: We studied DNM2 mRNA level in adults with B- and T-cell ALL, and found DNM2 is more highly expressed compared with normals in both forms of ALL. High DNM2 expression is significantly associated with poor overall survival (OS), high relapse rate, and leukaemia cell proliferation markers particularly in B-ALL. DNM2 expression is significantly higher in the patients with IKZF1 deletion compared to that of without deletion. Ikaros directly binds the DNM2 promoter in Nalm6 (B-ALL) and CEM (T-ALL) leukemic cells. Ikaros suppresses the transcription of DNM2 with luciferase reporter assay. Retroviral transduction of Ikaros results in the down-regulation of DNM2 in the leukemic cells. CK2 inhibitor, TBB increases Ikaros binding to promoter of DNM2 and suppresses DNM2 expression in an Ikaros-dependent manner in both leukemic cell lines and primary cells. TBB induced-increase of H3K9me3 binding on the promoter of DNM2 was also observed in leukemic cell lines and primary cells. Finally, DNM2 inhibitor-MiTMAB significantly suppresses the cell proliferation of Nalm6 and CEM cells with the WST-1 cell proliferation assay and has significantly synergistic effect with Ck2 inhibitor, CX-4945 in the cells. Conclusion: High DNM2 expression is associated with Ikarosdys-regulation, revealing their potential roles on the development of ALL. DNM2 inhibitor MiTMAB inhibits cell proliferation and has synergistic effect with CK2 inhibitor CX4945 in leukemic cells. Disclosures No relevant conflicts of interest to declare.

Background: Mixed-lineage leukemia (MLL) functions within the context of a large multiprotein complex including MLL, WDR5, RbBP5, and ASH2L that is required for maximal enzymatic activity. Each component is required for full histone H3 trimethyl Lysine 4 (H3K4me3) methyltransferase activity of the complex. The structure-function analysis shows that WDR5 mediates interaction between the MLL catalytic unit and the core complex, as well as the histone H3 substrate. Recently it is found that blocking MLL1-WDR5 interaction by an inhibitor could result in the complex disassembly, inhibition methyltransferase activity and suppression of proliferation of leukemia cells. Despite our growing knowledge about MLL1 fusion proteins and leukemia, very little is known about the role of WDR5 in leukemia. Methods: The WDR5 expression was determined by qPCR in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) patients. The genome-wide binding profiling of WDR5 and H3K4me3 was obtained by ChIP-seq. The effect of WDR5 on its target gene expression, cell proliferation and apoptosis was observed by qPCR, WST-1 cell proliferation assay and Annexin V-PE staining following the flow cytometry analysis, respectively, in leukemic cells with WDR5 shRNA knockdown Results: WDR5 expression is significantly increased in adult ALL and AML compared to that of normal bone marrow control. WDR5 high expression is associated with high risk factors in the patients. Also, its high expression is associated with MLL1 high expression; particularly the patients with WDR5 high expression plus MLL1 high expression has poor complete remission (CR) rate. We further identified the global genomic binding of WDR5 in RS4:11 ALL and THP-1 AML cells by ChIP-seq and detected more than 2000 binding peaks in the two leukemia cell lines. We also examined global H3K4me3 peaks, analyzed the correlation of WDR5 peaks with H3K4me3 peaks and found the genomic co-localization of WDR5 binding with H3K4me3 enrichment. Moreover, WDR5 knockdown by shRNA suppressed the cell proliferation, induced apoptosis, inhibited the expression of WDR5 targets on oncogenesis and anti-apoptosis, blocked the H3K4me3 enrichment on the promoter of these targets. We also observed the positive correlation of WDR5 expression with its targets in B-ALL and AML cohort. Conclusion: WDR5 has oncogenic effect and WDR5-mediated H3K4 methylation plays important role in the leukemogenesis Disclosures No relevant conflicts of interest to declare.

IKZF1 (Ikaros) encodes a DNA-binding protein that acts as a tumor suppressor in acute lymphoblastic leukemia. Deletion of one Ikaros allele results in the development of high-risk B-cell acute lymphoblastic leukemia (B-ALL) with a high incidence of relapse and poor prognosis. The mechanisms through which Ikaros suppresses leukemogenesis and that regulate Ikaros tumor suppressor activity in leukemia are unknown. Using a systems biology approach, we determined that Ikaros regulates transcription of genes that control two pathways that are crucial in leukemia cell proliferation: 1) cell cycle progression and 2) the phosphatidylinositol 3-kinase (PI3K) pathway. Gain- and loss-of-function experiments demonstrate that Ikaros represses the transcription of genes that promote cell cycle progression and the PI3K pathway and activates transcription of a gene that suppresses the PI3K pathway. We show that in high-risk B-ALL with deletion of one Ikaros allele, the function of Ikaros as a transcriptional regulator is impaired due to reduced DNA-binding affinity for promoters of its target genes. It has been shown that Ikaros DNA-binding affinity is regulated via direct phosphorylation by pro-oncogenic Casein Kinase II (CK2). CK2 is overexpressed in high-risk B-ALL as compared to normal B-cell precursors, which further reduces Ikaros function in high-risk B-ALL. Treatment of primary high-risk B-ALL (with deletion of one Ikaros allele) using the CK2 specific inhibitor, CX-4945, restored Ikaros function as a transcriptional regulator of the genes that regulate cell cycle progression and the PI3K pathway, and was associated with cell cycle arrest and loss of phosphorylation of the AKT kinase - a downstream target of the PI3K pathway. The use of serial quantitative chromatin immunoprecipitation (qChIP) analyses spanning the promoters of Ikaros target genes demonstrated that Ikaros can repress transcription of its target genes by two different mechanisms: 1) via recruitment of histone deacetylase 1 (HDAC1), which is associated with the formation of repressive chromatin characterized by H3K27me3 and loss of H3K9ac; and 2) via an HDAC1-independent mechanism which is associated with the formation of repressive chromatin characterized by H3K9me3, along with the loss of H3K9ac. The therapeutic effect of CK2 inhibition by CX-4945 on high-risk B-ALL was demonstrated in vivo using 4 different xenografts: 3 different high-risk primary pre-B-ALL xenografts and Nalm6 xenografts. Treatment with CX-4945 showed a strong therapeutic effect in all 4 xenografts, as evidence by reduced leukemia cell number in bone marrow and in spleen, along with prolonged survival of all xenografts. Expression analysis of Ikaros target genes that regulate cell cycle progression and the PI3K pathway in leukemia cells treated in vivo with CX-4945 revealed an expression pattern that was highly similar to that observed with Ikaros overexpression. This suggests that CK2 inhibition in vivo exerts its therapeutic effect on high-risk B-ALL via restoration of Ikaros function as transcriptional regulator of genes that promote cell cycle progression and the PI3K pathway. In summary, our results reveal that: 1) Ikaros functions as a tumor suppressor by suppressing cell cycle progression and the PI3K pathway; 2) Ikaros regulates transcription by inducing two distinct epigenetic alterations at promoters of its target genes and 3) CK2 inhibition with CX-4945 restores Ikaros function as a transcriptional regulator in vivo, and has a strong therapeutic effect in primary xenografts of high-risk B-ALL. These results provide support for the use of CK2 inhibitors in clinical trials for high-risk B-ALL. Supported by the National Institutes of Health R01 HL095120, and the Four Diamonds Fund Endowment. Disclosures No relevant conflicts of interest to declare.

Objective: T-cell acute lymphoblastic leukemia (T-ALL) is caused by collaboration of multiple genetic abnormalities in the transformation of T-cell progenitors. PHF6 is founded as a new key tumor suppressor and mutated in T-ALL. The clinical significance of PHF6 mutations has not been fully determined in adult T-ALL. This study aimed to screen the PHF6 mutations in adult T-ALL and explore the associations of PHF6 mutations with other genetic lesions, as well as their clinical relevance in adult T-ALL patients. Methods: We amplified the exons of PHF6, NOTCH1, FBXW7, PTEN and JAK1 following by DNA sequencing to identify the genomic mutations and examined the PHF6 mRNA level by qPCR in adult T-ALL patients. We also analyzed the correlations of PHF6 and NOTCH1 mutations with clinical features using a χ2 test and survival curve using the Kaplan-Meier method. Results: The 27.1% (16/59) PHF6 mutations including 10 novel mutations were detected in Chinese adult T-ALL. Six of 16 (37.5%) were frame-shift mutations, which could result in the deletion of the protein. We also observed PHF6 expression was significantly lower in T-ALL patients with PHF6 mutations compared with wide type cases (0.00423 vs. 0.06464, P=0.035) , indicating PHF6 mutations could be loss of function. Moreover, PHF6 mutation was significantly associated with NOTCH1 mutation(P=0.035). We further analyzed the domains involving co-existence mutations of NOTCH1 with PHF6. The most commonly mutated domains in NOTCH1 co-existed with PHF6 were HD-N only 6/12 (50.0%), followed by HD-C only 2/12(16.7%), PEST only 2/12(16.7%), HD-C+PEST 1/12(8.3%) and HD-N+HD-C 1/12(8.3%), indicating that HD domain (especially HD-N) of NOTCH1 may contribute to the synergistic effect on oncogenesis of the two genes. Furthermore, the patients with co-existence of PHF6 and NOTCH1 mutations had lower hemoglobin and higher incidence of splenomegaly or lymphadenopathy compared to that without co-existence of the mutations (95.0 vs 122.0, P=0.007; 81.8% vs 38.3%, P=0.009; 90.9% vs 44.7%, P=0.006). Importantly, the patients with co-existence of mutations in PHF6 and NOTCH1 (PHF6mutNOTCH1mut) had significant shorter event-free survival (EFS) compared with that without co-existence (non-PHF6mutNOTCH1mut)(2.0 months vs. 12.0 months, P=0.027). Conclusion: PHF6 is inactivated in T-ALL due to its low expression and mutations. PHF6 mutation is co-existed with NOTCH1 mutations, and the patients with PHF6mutNOTCH1mut had a poor prognosis. Our results indicated synergistic effect of PHF6 and NOTCH1 mutations on leukemogenesis and PHF6mutNOTCH1mut may be potential prognostic marker in adult T-ALL. Disclosures No relevant conflicts of interest to declare.

Background Epigenetic changes in DNA and chromatin are regarded as emerging major players for hematopoietic stem cells development and lineage differentiation. Epigenetic deregulation of gene expression leads to leukemia and reversibility of epigenetic modifications makes DNA and chromatin changes attractive targets for therapeutic intervention. The promyelocytic leukemia HL-60 cell can differentiate into microphage, granulocyte and monocyte with stimulation of phorbol myristic acid (PMA), dimethylsulfoxide (DMSO), and Vitamin D3, respectively, by affecting the gene expression on cell cycle and cell differentiation. However, how epigenetic regulation on gene expression in the inducible differentiation is still undetermined. Methods and Results We did the microarray analysis to identify the genome-wide gene expression profile of HL-60 cells in various time points(0h, 6h, 1d,2d,4d and 6d) under the treatment of PMA, DMSO and D3 and found that around 3000 genes are significantly altered commonly in the cells upon the 3 treatments. The percentage of down-regulated genes in the all commonly altered genes is significantly higher than that of up-regulated genes, and the significantly altered genes showed significantly physical clustering on chromosome loci, indicating the epigenetic regulation involved in the regulation of the gene expression. We did observed the expression changes of epigenetic enzymes in the process and further did the ChIP-on-ChIP analysis by using the customer array tiling the selected changed genes and 5 genomic loci hybridizing with ChIP’d DNA of histone markers (H3K4me3, H3K9me3 and H3K27me3). We found that the obvious decrease of H3K4me3 binding and increase of H3K9me3 and H3K27me3 are observed in the promoter of the commonly down-regulated genes. These data indicated histone H3K4, H3K9 and H3K27 marker play important roles in shaping the chromatin state and regulating gene expression. Conclusion We identified the signature genes controlling the inducible differentiation of promyelocytic leukemiacells and found the epigenetic mechanism regulating the gene expression in the process. Disclosures No relevant conflicts of interest to declare.