ALBERT

Description: The primary cause of treatment failures in acute myeloid leukemia is usually associated with defects in the apoptotic pathway. Several studies suggest that 2-(4-aminophenyl)-7-methoxybenzothiazole (7-OMe-APBT) may potentially induce apoptosis of cancer cells. Thus, the present study was conducted to explore the cytotoxic effect of 7-OMe-APBT on human leukemia U937 cells. The apoptosis of human leukemia U937 cells induced by 7-OMe-APBT was characterized by an increase in mitochondrial membrane depolarization, procaspase-8 degradation, and tBid production. Down-regulation of FADD blocked 7-OMe-APBT-induced procaspase-8 degradation and rescued the viability of 7-OMe-APBT-treated cells, suggesting the involvement of a death receptor-mediated pathway in 7-OMe-APBT-induced cell death. Increased TNF-α expression, TNFR2 expression, and p38 MAPK phosphorylation were noted in 7-OMe-APBT-treated cells. Pretreatment with a p38 MAPK inhibitor abolished 7-OMe-APBT-induced TNF-α and TNFR2 up-regulation. 7-OMe-APBT stimulated p38 MAPK/c-Jun-mediated transcriptional up-regulation of TNFR2, while the increased TNF-α mRNA stability led to TNF-α up-regulation in 7-OMe-APBT-treated cells. Treatment with 7-OMe-APBT up-regulated protein phosphatase 2A catalytic subunit α (PP2Acα) expression via the p38 MAPK/c-Jun/ATF-2 pathway, which, in turn, promoted tristetraprolin (TTP) degradation. Pretreatment with a protein phosphatase 2A inhibitor or TTP over-expression abrogated TNF-α up-regulation in 7-OMe-APBT-treated cells. Abolishment of TNF-α up-regulation or knock-down of TNFR1/TNFR2 by siRNA restored the viability of 7-OMe-APBT-treated cells. Taken together, our data indicate a connection between p38 MAPK-mediated TNF-α and TNFR2 up-regulation and 7-OMe-APBT-induced TNF-α-mediated death pathway activation in U937 cells. The same pathway also elucidates the mechanism underlying 7-OMe-APBT-induced death of human leukemia HL-60 cells. This article is protected by copyright. All rights reserved

Description: Key Points A comprehensive study of 19 gene mutations and their cooperation, including the first report of ASXL1 and TET2 mutations in pediatric AML. The development of pediatric AML requires fewer gene mutations than adult AML.

Description: Abstract 2393 Somatic mutations of ASXL1 gene have been described in patients with myeloid malignancies and were associated with inferior outcomes. ASXL1 mutations have also been detected in myeloid blast crisis of chronic myeloid leukemia (CML) patients. The mechanisms of acute myeloid leukemia (AML) transformation and functional role of ASXL1 mutations in the leukemogenesis remain to be determined. Recently, we identified PHD domain deletion mutations (R693X and L885X) in patients with CML in myeloid blast crisis and/or AML with minimal differentiation (M0). In the present study, we aimed to investigate the role of PHD domain deletion mutations in the pathogenesis of AML transformation. The K562 cells carrying Philadelphia chromosome, serves as a model to study the molecular mechanisms associated with leukemogenesis. Our result showed that R693X/L885X mutations inhibited PMA-treated megakaryocytic differentiation with the change of physiological characteristic features and suppressed the induction of CD61, a specific cell surface marker of megakaryocytes. We also found that FOSB, a member of Fos family of AP-1 transcription factors was down-regulated in K562 cells expressing R693X and L885X compared to wild-type ASXL1 during PMA-mediated megakaryocytic differentiation. Examination of intracellular signaling pathways showed that the mutant ASXL1 protein prevented PMA-induced megakaryocytic differentiation through the inactivation of ERK, AKT and STAT5 which are required for differentiation. Further, ASXL1 depletion by shRNA in K562 cells led to enhanced cell proliferation, increased colony formation and impaired PMA-mediated differentiation. Previous studies in Drosophila had revealed that Asxl forms the protein complexes of both Trithorax and Polycomb groups that are required for maintaining chromatin in both activated and repressed transcriptional states. By using Western blot analysis, we demonstrated that PHD domain deletion mutations of ASXL1 significantly suppressed the transcriptionally repressive mark H3K27 trimethylation, however no effect on methylated H3K4 (H3K4me2 and H3K4me3), an active histone mark in K562 cells. Co-immunoprecipitation analysis revealed that wild-type, but not PHD domain deletion mutations of ASXL1 interact with EZH2, a member of the polycomb repressive complex 2 (PRC2). Importantly, PHD deletion mutations or downregulation of ASXL1 resulted in the suppression of EZH2 in K562 cells. Our study demonstrated that PHD deletion mutations of ASXL1 resulted in a loss-of-function which exhibited direct effects on the proliferation and differentiation and also proposed a specific role for ASXL1 in epigenetic regulation of gene expression in K562 cells. Disclosures: No relevant conflicts of interest to declare.

Description: Background Chronic myeloid leukemia (CML) is characterized by the BCR-ABL1 fusion gene. Despite the dramatic improvement of its prognosis in recent years by the development of tyrosine kinase inhibitors (TKIs), a minority of chronic phase (CP) CML patients fail to respond to TKI therapies and progress to blast crisis (BC), showing dismal clinical outcomes. While acquired mutations in ABL1 kinase have been identified as a common mechanism for TKI resistance, recent genetic studies have revealed that patients with BC frequently harbor one or more genetic alterations implicated in myeloid malignancies, suggesting additional mutations other than ABL1 mutations might drive disease progression. However, our knowledge about the mechanism of TKI resistance and progression to BC is largely limited by the scarcity of matched CP and BC samples, which were investigated for genetic alterations in relatively small number of genes. Here, we performed comprehensive genomic studies of CML-BC using paired CP and BC samples to investigate the mutation profiles associated with BC. Method We performed whole-exome sequencing of 53 patients with CML-BC, including 40 myeloid and 13 lymphoid crisis cases, as well as corresponding CP controls to investigate acquired mutations during disease progression from CP to BC. We also performed targeted-capture sequencing of known and putative driver genes in an additional 15 CML-BC samples. Combined, a total of 68 CML-BC samples were analyzed for somatic mutations, copy number abnormalities, and structural variations. Results Commonly affecting ASXL1, GATA2, and IKZF1, mutations were found only in a minority of CP cases (10/53 [19%]). However, most cases acquired somatic mutations during disease evolution from CP to BC; in whole-exome sequencing, an average of 17 additional non-synonymous mutations were newly acquired per case during evolution from CP to BC. Mutations in CML-BC frequently involved known driver genes, such as ASXL1, RUNX1, ABL1, TP53, BCOR/BCORL1, and WT1. In addition, we identified novel targets of recurrent mutations, including UBE2A, NBEAL2 and KLC2. Of note, most these driver mutations were not detected in corresponding CP samples and newly acquired, whereas ASXL1 mutations were often found in corresponding CP samples in a minor population, suggesting that ASXL1 mutations at CP might play an important role in the disease progression to BC. Mutational profiles were similar between cases with and without a history of TKI therapy before BC, except for frequent ABL1 mutations among TKI-treated cases, mostly affecting the kinase domain. Compared with lymphoid BC, myeloid BC showed a higher number of somatic mutations, which was more prominent for ASXL1, TP53, and WT1 mutations. Copy number abnormalities were rarely found in CML-CP cases (8/53), but were common and newly acquired in 29 (55%) cases with CML-BC, 18 of which showed complex karyotype-like (≥3) abnormalities. Amplification of chromosome 6 and/or 8 were characteristics of myeloid BC, while deletion of chromosome 7 was more characteristic of lymphoid BC. In some cases, structural variations other than BCR-ABL1 translocation were newly acquired in CML-BC, which frequently involved genes implicated in myeloid malignancies such as RUNX1, CBFB, and MECOM. When mutations, copy number abnormalities, and structural variants were combined, most BC cases had at least one driver alterations, which might be involved in CML-BC. Conclusion Through a comprehensive sequencing analysis using paired samples of CP and BC, we demonstrate a role of additional driver events during the clonal evolution to BC. Additional mutations were common even in CML-CP, some of which might contribute to the progression to BC. Disclosures Takaori-Kondo: Celgene: Honoraria, Research Funding; Novartis: Honoraria; Janssen Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria.

Description: Introduction: IKZF1 deletion was firstly reported to be very frequent in Philadelphia (Ph) positive B-cell acute lymphoblastic leukemia (B-ALL), and continuously found in other B-ALL subtypes. IKZF1plus defined as the presence of any of CDKN2B, CDKN2A,PAX5 or PAR1 in the absence of ERG deletion and was reported to describe a new minimal residual disease (MRD)-dependent poor prognostic profile in B-ALL.1 We aimed to analyze the frequency and prognostic relevance of IKZF1 deletion with or without co-occurring gene alterations in Taiwanese children with B-ALL treated with TPOG-ALL-2002 protocol or MRD-directed TPOG-ALL-2013 protocol. Methods: Bone marrow samples at diagnosis from 561 children excluding Ph+ and infant ALL were analyzed. Detection of gene deletion was carried out with multiplex ligation dependent probe amplification (MLPA) kit (SALSA MLPA P335 and P327) and mutations of RAS pathway genes (NRAS, KRAS, and PTPN11) were assessed by Sanger sequencing. The outcome was analyzed on 259 patients treated with TPOG-ALL-2002 protocol and 158 patients treated with TPOG-ALL-2013 protocol. Results: IKZF1 deletions were present in 11.2% of 561 patients. DEL 4-7, DEL 1-8, and other variants were present in 50.8%, 14.3%, and 34.9%, respectively of 63 IKZF1-deleted patients. Co-existence of additional deletions with IKZF1 deletion was detected in 34.0% for CDKN2B, 37.3% for CDKN2A, 27.7% for PAX5, 2.1% forPAR1, and 8.0% for ERG. IKZF1plus comprised of 4.4% of 546 patients who had all the genes examined. Co-existed mutations of RAS pathway genes were detected in 5.0% for PTPN11, 13.3% for NRAS, and 10.0% for KRASin the IKZF1-deleted patients. The 5-year event-free survival (EFS) of IKZF1-undeleted patients was significantly better compared with IKZF1-deleted patients in TPOG-2002 cohort (82.5 ± 2.6% vs. 56.9 ± 9.1%, P = 0.002) as did TPOG- 2013 cohort (89.4 ± 3.9% vs. 59.1 ± 19.8%, P = 0.012). The 5-year overall survival (OS) of IKZF1-deleted patients was worse than that of patients with undeleted IKZF1 in TPOG-2013 cohort (58.3 ± 19.8% vs. 89.2 ± 4.2%, P = 0.004) but not in TPOG-2002 cohort (90.9 ± 5.0% vs. 86.9 ± 2.3%, P = 0.846). DEL 1-8 patients had an inferior 5-year EFS (33.3 ± 19.2%) compared with DEL 4-7 (63.3 ± 12.0%,) or DEL-others (60.0 ± 18.4 %) in TPOG- 2002 cohort (P = 0.003). No significant difference in OS was observed between patients among different IKZF1-deleted subtypes in TPOG-2002 cohort (P = 0.283) (Fig. 1A) but there was different in TPOG-2013 cohort (P = 0.003) (Fig. 1B). Patients with IKZF1 deletion alone had comparable 5-year EFS and 5-year OS compared with patients with IKZF1plus in TPOG-2002 cohort whereas patients with IKZF1 deletion alone had worse 5-year EFS (33.3 ± 27.2% vs. 80.0 ± 17.9%, P = 0.284) and 5-year OS (33.3 ± 27.2% vs. 80.0 ± 17.9%, P = 0.415) than that of KZF1plus in TPOG-2013 cohort. The prognostic impact of IKZF1 deletion with co-existed gene alterations in TPOG-2002 cohort was further analyzed as follows: IKZF1plus with ≧ 2 co-existed deletions had an inferior 10-year EFS (23.8 ± 20.3%) compared with patients with IKZF1 deletion alone (36.7 ± 17.5%) or IKZF1-undeleted patients (81.5 ± 2.7%) (P = 0.005) (Fig. 1C). Three patients carried both IKZF1 and ERG deletions had a superior 5-year EFS (100%) compared with IKZF1-deleted alone or IKZF1-undeleted patients (P = 0.005) (Fig. 1D). The 10-year EFS of patients with any gene mutation of RAS pathway was worse than that of patients with RAS wild-type genes (66.6% ± 6.4% vs. 83.7% ± 2.6%, P = 0.011). In multivariate analysis, in addition to the initial white blood count 〉 50 x 109/L and KMT2A-rearranged, IKZF1 deletion (HR=2.609, 95% CI: 1.363-5.034; P = 0.004) and RAS pathway gene mutations (HR=2.360, 95% CI: 1.336-4.168; P = 0.003) were independent genetic predictors for inferior EFS. Co-existence of IKZF1 deletion with RAS pathway mutations had worst 5-year EFS (20.0% ± 12.6%, P 〈 0.0001, Fig. 1E), and 10-year OS (53.3% ± 17.6%, P = 0.042, Fig. 1F). Conclusions: Our results showed that IKZF1 deletion alone was associated with an inferior OS in MRD-directed TPOG-2013 treated cohort but not in TPOG-2002 cohort in which it became significant when co-existed with RAS pathway mutations. Reference: 1. IKZF1plus defines a new minimal residual disease-dependent very-poor prognostic profile in pediatric B-cell precursor acute lymphoblastic leukemia. J Clin Oncol. 2018, 1240-1249. Figure 1 Disclosures No relevant conflicts of interest to declare.

Description: Background Chronic myeloid leukemia (CML) is characterized by the BCR-ABL1 fusion gene. Despite the use of tyrosine kinase inhibitors (TKIs), a minority of chronic phase (CP) CML patients fail TKI therapies and progress to blast crisis (BC), showing dismal outcomes. Although genetic studies have revealed that CML-BC frequently carries not only ABL1 mutations but other driver mutations, our knowledge about the mechanism of TKI resistance and progression to BC is still limited by a relatively small number of patients and/or genes analyzed in each study. Moreover, it remains elusive whether mutations can predict clinical outcomes of BC patients, in which few biomarkers are known. Here, we investigated a large cohort of CML patients to reveal the landscape of genetic lesions and those predicting clinical outcomes in CML. Methods We performed whole-exome sequencing (WES) of paired CP and BC samples from 52 patients and targeted-capture sequencing that covered myeloid driver genes in 32 BC and 19 CP samples. Combined with public WES data for 24 BC and 77 CP, we analyzed a total of 108 BC and 148 CP samples. Results In WES analysis of paired CP and BC samples, an average of 5.3 nonsynonymous mutations were acquired during disease progression from CP to BC. Notably, a Poisson regression model revealed that the number of acquired mutations was positively correlated with time to progression from CP to BC (P 〈 0.001) and negatively with TKI therapy after CP diagnosis (P = 0.0093), although the correlation of the number of driver mutations in CML-BC with time to progression was not clear. These results suggest that the use of TKI effectively reduces the size of tumor populations at risk for clonal evolution by acquiring random mutations, by which prevents BC. In CML-BC, we found frequent mutations not only in known mutational targets in other hematological malignancies, such as RUNX1, ABL1, ASXL1, BCOR/BCORL1, TP53, and WT1, but also in other genes recently reported in BC (UBE2A and SETD1B) and previously unreported mutational targets in cancer (KLC2 and NBEAL2). Deep amplicon-sequencing revealed that ASXL1 mutations were already present at the time of CP diagnosis in most cases, whereas others such as RUNX1, ABL1, and TP53 mutations were absent in CP and newly emerged during progression to BC. Some abnormalities, such as +21, +8, and ASXL1 mutations, were more enriched in myeloid than lymphoid crisis, while others, including CDKN2A/B and IKZF1 deletions, -7/del(7p), -9/del(9p), and ABL1 mutations, vice versa. By contrast, abnormalities such as RUNX1 mutations and double Ph were almost equally observed in both crises. In univariate analysis of clinical factors for overall survival (OS) in 77 CML-BC cases for whom survival information was available, TKI-containing therapy for BC was significantly associated with a better OS, whereas genetic lesions including ASXL1 and TP53 mutations, del(17p), amp(17q), +19, and +21 had a negative impact on OS. Conspicuously, patients with TP53 mutations, del(17p), and amp(17q) showed an especially dismal outcome. We then performed a multivariate analysis using a Cox proportional hazard regression model, focusing on 36 TKI-treated patients, because TKI-containing therapy has been shown to improve OS and therefore, is a standard choice of therapy. We found that ASXL1 and BCOR mutations, complex copy-number alterations, amp(17q), and +21 were independent predictors for worse prognosis. Based on the number of these unfavorable factors, patients were classified into three subgroups showing distinct prognosis, where the 2-year OS rate was 71.8%, 15.6%, and 0% for patients with 0, 1, and ≥2 risk factors, respectively (P 〈 0.001). Finally, we explored the genetic abnormalities and clinical outcomes in CML-CP. In CP, only ASXL1 was mutated at a frequency comparable to that in BC, while others, including TET2, KMT2D, PTPN11, RUNX1, and WT1, were mutated at much lower frequencies. Of interest, patients who later developed BC more frequently had at least one genetic abnormality, suggesting that mutations found at the time of CP might play a role in driving CML cells to BC under the pressure of TKI treatment. Conclusion Our study clarified a comprehensive registry of genetic lesions in BC in a large cohort of CML patients and their prognostic impact, which should provide a clue to the development of better therapy/management for patients with CML. Disclosures Takaori-Kondo: Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Astellas Pharma: Honoraria, Research Funding; Ono Pharmaceutical: Research Funding; Thyas Co. Ltd.: Research Funding; Takeda: Research Funding; CHUGAI: Research Funding; Eisai: Research Funding; Nippon Shinyaku: Research Funding; Otsuka Pharmaceutical: Research Funding; Pfizer: Research Funding; OHARA Pharmaceutical: Research Funding; Sanofi: Research Funding; Novartis Pharma: Honoraria; MSD: Honoraria. Mitani:CHUGAI: Research Funding; Takeda: Research Funding; KYOWA KIRIN: Consultancy, Research Funding. Ogawa:Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Eisai Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Shih:Novartis: Research Funding; Celgene: Research Funding; PharmaEssentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees.