Objective Internal tandem duplication (ITD) of fms-like tyrosine kinase 3 (FLT3) is one of the most common mutations in acute myeloid leukemia (AML), occurring in nearly 30% of cases. FLT3-ITD involves in-frame duplication of 3-400 base-pairs at the juxta-membrane domain, resulting in ligand-independent activation of FLT3 signaling. Downstream effectors include activation of ERK/STAT5 via SRC kinase, activation of PI3K/AKT, phosphorylation of FOXO3A, down-regulation of equilibrative nucleoside transporter 1 (ENT1) for cytarabine, and induction of reactive oxygen species (ROS) that may lead to increased DNA damage and defective repair. The present study investigated if the latter can be effectively targeted for the treatment of this AML subtype. Methods Primary samples from patients with FLT3-ITD AML, human cell line carrying FLT3-ITD (MOLM-13) as well as mouse B-lymphoid Ba/F3 cells transduced with human FLT3-ITD were used in this study. Traffic Light Reporter (TLR) assay was used to measure fidelity of double-strand breaks (DSB) repair, either via error-free homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Percentage of repair events by HR or NHEJ were quantified by flow cytometry. DNA DSB were examined by γ-H2AX foci staining using confocal microscopy. Single-cell DSB were quantified by neutral comet assay and analysed by OpenComet software. The tail moment was calculated as the length of comet tail multiplied by the tail DNA %. MOLM-13 was transplanted into NOD/SCID/IL2Rg-/- (NSG) mice by tail vein injection. Treatment comprised cytarabine (25mg/kg, i.p., day 1-5) and doxorubicin (1.5mg/kg, i.v., day 1-3), with or without olaparib (25mg/kg, i.p., day 1-5). Comparisons between groups of numerical data were evaluated using Student's t-test. P-values less than 0.05 were considered statistically significant. Results To investigate the link between FLT3-ITD AML and DNA damage response (DDR), expression of critical DDR genes in primary AML samples was examined by real-time quantitative PCR. The panel of genes included apical kinase ATM, ATR and DNA-PKcs; DNA damage mediators BRCA1,BRCA2 and PARP1; downstream response kinase CHEK1 and CHEK2 and effectors TP53. Among them, BRCA2 was significantly down-regulated in FLT3-ITD AML(Wild-type FLT3=18 samples; FLT3-ITD=13 samples; Average dCt 9.7 in WT vs. 10.6 in ITD; p<0.05). Down-regulation of BRCA2 in FLT3-ITD AML was further validated in a microarray database GSE15434 from a multicenter study investigating gene expression profiles of normal karyotype AML( FLT3-WT=148; FL3-ITD=86; ; log2 gene expression 5.61 in WT vs 5.45 in ITD; p<0.001). As BRCA2 is an important protein mediating HR, a DSB DNA repair TLR assay was performed. HR was significantly down-regulated in Ba/F3-FLT3-ITD as compared with parental Ba/F3 line by flow cytometry(1.21% HR repair in control vs. 0.44% in ITD; p<0.05) while NHEJ was unaffected (1.90% NHEJ repair in control vs. 2.38% in ITD; p=0.50). Down-regulation of BRCA2 expression and defective HR in FLT3-ITD AML was reminiscent of BRCA mutant breast and ovarian cancers. Therefore, the effects of poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib were examined. Ba/F3-FLT3-ITD were more sensitive to olaparib than parental line. Olaparib inhibited base excision repair and increased DSB as indicated by increased number of γ-H2AX foci and increased tail moment in Ba/F3-FLT3-ITD cells when compared to parental line. Olaparib-induced DSB in Ba/F3-FLT3-ITD cells was mainly repaired by NHEJ as shown by colocalization of γ-H2AX foci with 53BP1. Combination of chemotherapy (cytarabine and doxorubicin) and olaparib synergistically reduced leukemic cell growth of MOLM-13 in NSG murine xenograft model. Conclusion Results from the present study supported the use of PARPi in the treatment of FLT3-ITD AML. Its therapeutic benefits in combination with chemotherapy would have to be further evaluated. Acknowledgements: Health and Medical Research Fund (Project number: 04152326) and Li Shu Fan Medical Foundation, LKS Faculty of Medicine, University of Hong Kong. Disclosures No relevant conflicts of interest to declare.
Objectives: Acute myeloid leukemia (AML) carrying internal tandem duplication (ITD) of Fms-Like Tyrosine Kinase 3 (FLT3) is associated with high relapse risk and adverse outcome. This single-arm, phase 2 study evaluated efficacy and safety of combination treatment with FLT3 inhibitor quizartinib and protein translation inhibitor omacetaxine mepesuccinate (OME), referred herein QUIZOM - in elderly patients with newly diagnosed FLT3-ITD AML or young patients with relapsed/refractory (R/R) diseases, whose outcome is hitherto dismal. Methods: R/R FLT3-ITD AML patients ≥ 18 years old or newly diagnosed patients ≥ 65 years old were recruited. Treatment comprised quizartinib 30 mg daily and OME [2 mg daily for 7 (first course) or 5 days (second course onwards) every 21-28 days] until disease progression or allogeneic hematopoietic stem cell transplantation (HSCT). Quizartinib was given as post HSCT maintenance after engraftment at doses ranging from 30 mg twice weekly to 30 mg daily, depending on blood counts. Primary endpoint was composite complete remission (CRc) defined by CR+CRi (CR with incomplete haematological recovery). Secondary endpoints were leukaemia-free (LFS) and overall survival (OS). Molecular responses were evaluated by FLT3-ITD based on PCR and NPM1 variant allele frequency (VAF) based on digital PCR (ddPCR). Mutation profiling was performed by MiSeq Next Generation Sequencing (NGS) based on IDT xGen Lockdown probes custom panel of 67 genes or TruSight myeloid panel of 54 genes. Results: Twenty-nine patients (R/R case=22; newly diagnosed case=7) were recruited between November 2017 and July 2019. Their clinicopathologic characteristics were shown in Table 1. Twenty-seven patients completed at least one course of QUIZOM (median= 2 courses, range: 1-20 courses) of whom 22 (R/R=19; newly diagnosed=3) achieved CR/CRi [CR=2 (7%); CRi=20 (74%), CRc=22 (81%)], 3 patients showed partial remission (PR) and 2 patients did not respond. All 6 patients who had prior FLT3 inhibitors (sorafenib or midostaurin) achieved CR/CRi. Median LFS and OS of CR/CRi group were 5.2 and 11.07 months (Figure 1). Seven patients received allogeneic HSCT of whom all have remained in remission post HSCT as of 31 July 2019 (OS 6.43 to 19.8 months) (Figure 1). Deep molecular responses (DMR) as defined by FLT3-ITD VAF ≤0.1% and NPM1 VAF ≤ 0.001% could be accomplished in 78% and 27% patients. Adverse effects associated with QUIZOM were primarily haematological and non-haematologic adverse effects were scarce. Two patients succumbed before commencement and on day 1 of QUIZOM due to pneumonia and intracranial haemorrhage. None of recurrently mutated genes was significantly associated with treatment responses in this cohort. Conclusion: QUIZOM is effective and safe for newly diagnosed and R/R FLT3-ITD AML. Acknowledgements: Li Shu Fan Medical Foundation, S.K. Yee Medical Foundation, Croucher Foundation, LKS Faculty of Medicine, University of Hong Kong, Daiichi Sankyo Inc. Disclosures No relevant conflicts of interest to declare.
One of the greatest challenges in acute myeloid leukemia (AML) treatment is preventing relapse. Leukemia cells can hide in bone marrow niche or vascular niche. Hence, many chemical drugs cannot kill these cells. To characterize migration and adhesion properties of leukemia cells in specific niches, CXCR4/SDF- 1α signal pathway has been widely used for investigation. AMD3100 is treated as one of the most common chemical drugs that can inhibit this signal. In the current study, we particularly investigate the effect of AMD3100 on the adhesion property of leukemia cells on stromal cells by using engineering tools, namely, optical tweezers (OT) and dielectrophoresis (DEP), to probe single cell property. AMD3100 not only inhibits the CXCR4/SDF- 1α signal pathway but also reduces gene expression of CXCR4 and VLA-4 on leukemia cells. The drug also softens leukemia cells. This work provides a new way to investigate cell behavior under drug treatment. The use of combined engineering tools will benefit drug discovery and assessment for leukemia treatment.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.