Ying-Jung Huang

Cooperating gene mutations in childhood acute myeloid leukemia with special reference on mutations of ASXL1, TET2, IDH1, IDH2, and DNMT3A

Gene mutations involving epigenetic regulators recently have been described in adult acute myeloid leukemia (AML). Similar studies are limited in children. We analyzed gene mutations and cooperation in pediatric AML with special reference on mutated epigenetic regulators. Nineteen gene mutations, including 8 class I genes, 4 class II genes, WT1 and TP53 (class III), and 5 epigenetic regulator genes (class IV), were analyzed in 206 children with de novo AML. Mutational analysis was performed with polymerase chain reaction-based assay followed by direct sequencing. One hundred seventeen of 206 patients (56.8%) had at least one mutation: 51% class I, 13% class II, 6.8% class III, and 5.6% class IV. FLT3-internal tandem duplication was most frequent, and 29% of patients had more than one gene mutation. Two patients carried ASXL1 mutations, both with t(8;21), 2 had DNMT3A mutations, 2 had IDH1 mutations, 1 had IDH2 mutation, and 3 had TET2 mutations. Both patients with IDH1 mutations had AML-M0 subtype and MLL-partial tandem duplication. Cooperating mutations with mutated epigenetic regulators were observed in 8 of 10 patients. We conclude that mutated epigenetic regulators were much less than those in adult AML but with frequent cooperating mutations. ASXL1, TET2, and IDH1 mutations were associated with specific genetic subtypes.

Biological Activities of RUNX1 Mutants Predict Secondary Acute Leukemia Transformation from Chronic Myelomonocytic Leukemia and Myelodysplastic Syndromes

Purpose: Transcription factor RUNX1 is essential for normal hematopoiesis. High mutation frequencies of RUNX1 gene in chronic myelomonocytic leukemia (CMML) and myelodysplastic syndromes (MDS) have been described, whereas the biologic significances of the mutations were not investigated. Here, we aimed to correlate the biologic activities of the RUNX1 mutants with the clinical outcomes of patients. Experimental Design: We examined the mutational status of RUNX1 in 143 MDS and 84 CMML patients. Then, we studied the DNA and CBFβ binding abilities of all the RUNX1 mutants identified by using electrophoretic mobility shift assay and co-immunoprecipitation assay, and also determined their activities on target C-FMS gene induction by Western blotting and luciferase reporter assay. Using luciferase reporter assay, the relative biologic activities of each RUNX1 mutant could be quantified and correlated with the patient outcomes by statistical analyses. Results: We observed that most RUNX1 mutants had reduced abilities in DNA binding, CBFβ heterodimerization, and C-FMS gene induction. The relative biologic activities of RUNX1 mutants were grouped into high- and low-activity mutations. Correlation of the activities of RUNX1 mutants with the clinical outcomes revealed that patients harboring lower activities of RUNX1 mutants had a higher risk and shorter time to secondary acute myeloid leukemia transformation in MDS and CMML. In multivariate analysis, low RUNX1 activity remained an independent predictor for secondary acute myeloid leukemia–free survival in MDS patients. Conclusions: The biologic activity rather than the mutational status of RUNX1 might be an indicator in predicting outcome of patients with MDS and CMML. Clin Cancer Res; 21(15); 3541–51. ©2015 AACR.

Involvement of Gpr125 in the myeloid sarcoma formation induced by cooperating MLL/AF10(OM‐LZ) and oncogenic KRAS in a mouse bone marrow transplantation model

Oncogenic N‐/KRAS mutations were frequently associated with MLL/AF10 in acute myeloid leukemia with myeloid sarcoma (MS). To study the cooperating leukemogenesis by MLL/AF10 and KRAS mutation, we retrovirally transduced MLL/AF10(OM‐LZ) and KRASG12C into mouse bone marrow cells and generated two immortalized cell lines. The cells carrying cooperating MLL/AF10(OM‐LZ) and KRASG12C had immature myelomonocytic phenotypes. Compared to a previously established cell line carrying MLL/AF10(OM‐LZ) alone, cooperation of MLL/AF10(OM‐LZ) with KRASG12C blocked the cells at a more immature myelomonocytic stage with reduced expression of monocyte/macrophage markers. The mice transplanted with the cells carrying cooperating MLL/AF10(OM‐LZ) and KRASG12C, liked those transplanted with the cells carrying MLL/AF10(OM‐LZ) alone, induced myeloproliferative disease‐like myeloid leukemia, but in a shorter latency and formed multiple MS at the adipose tissues of skin, peritoneum and intraperitoneal cavity. Cooperation of MLL/AF10(OM‐LZ) with KRASG12C increased cell adhesion via upregulation of an adhesion G‐protein‐coupled receptor Gpr125. Knockdown of Gpr125 in the cells by short hairpin RNA reduced cell aggregation and diminished MS formation in the transplanted mice. Our results indicated that upregulation of Gpr125 by cooperating MLL/AF10(OM‐LZ) and KRASG12C promoted cell adhesion and contributed to the MS formation.

Cooperation of MLL/AF10(OM‐LZ) with PTPN11 activating mutation induced monocytic leukemia with a shorter latency in a mouse bone marrow transplantation model

PTPN11 mutation, a RAS signaling pathway mutation, is associated with MLL translocations in acute leukemia. A girl with MLL/AF10 AML was found to carry PTPN11G503A. To study the impact of PTPN11G503A cooperating with MLL/AF10 on leukemogenesis, we established a retroviral transduction/transplantation mouse model. Compared to the MLL/AF10(OM‐LZ) leukemia cells harboring PTPN11wt, the cells harboring PTPN11G503A were hypersensitive to GM‐CSF and IL3, and more resistant to death upon treatment with daunorubicin but sensitive to cytarabine. The cells harboring PTPN11G503A autonomously differentiated into macrophages (1.8%) in the medium containing IL3. Further studies showed that the cells had an elevated (∼2.9‐fold) Csf1 transcription level and secreted more (∼4.5‐fold) M‐CSF to the medium which can stimulate monocyte/macrophage differentiation of BM cells. Mice transplanted with the cells harboring PTPN11G503A had a higher concentration of M‐CSF in plasma. When mixed with the MLL/AF10(OM‐LZ) leukemia cells harboring PTPN11wt, the cells harboring PTPN11G503A had an increased competitive engraftment and clonal expansion in the BM and spleen of recipient mice, although no competitive growth advantage was observed in the in vitro co‐culturing assays. The mice transplanted with the MLL/AF10(OM‐LZ) cells harboring PTPN11wt developed myelomonocytic leukemia, while those transplanted with the cells harboring PTPN11G503A‐induced monocytic leukemia in a shorter latency. Our results demonstrated that addition of PTPN11G503A to MLL/AF10 affected cell proliferation, chemo‐resistance, differentiation, in vivo BM recruitment/clonal expansion and accelerated disease progression.

Clinical and biological relevance of genetic alterations in pediatric T-cell acute lymphoblastic leukemia in Taiwan

The leukemogenesis of T‐cell acute lymphoblastic leukemia (T‐ALL) involves multistep processes of genetic alterations. We aimed to determine the genetic alterations including common fusion transcripts, overexpression of T‐cell transcription factor oncogenes, and deletion or mutation of targeted genes in pediatric T‐ALL in Taiwan as well as their impact on outcomes in those treated with the Taiwan Pediatric Oncology Group‐ALL‐2002 protocol.

Genetic and Epigenetic Perturbations by DNMT3A-R882 Mutants Impaired Apoptosis through Augmentation of PRDX2 in Myeloid Leukemia Cells

DNA methyltransferase 3A (DNMT3A) is mutated in various myeloid neoplasms including acute myeloid leukemia (AML), especially at the Arg882 and associated with inferior outcomes. Here, we report that the DNMT3A-Arg882His/Cys (R882H/C) mutations led to inactivation of apoptosis through DNA damage signaling following the impairment of differentiation of myeloid leukemia cells. Gene expression profiling analysis revealed aberrant expression of several cell-cycle and apoptosis-related genes, and the DNA methylation assay identified both hypo- and hypermethylation features in different regions throughout the whole genome of DNMT3A mutants-transduced myeloid cells. We found that DNMT3A-R882H/C mutations upregulated the expression of an antioxidant protein, pyroxiredoxin-2 (PRDX2), at the mRNA and protein levels with decreased accumulation of reactive oxygen species (ROS). Augmentation of ROS generation by ROS accumulating agent or by knockdown of PRDX2 from myeloid cells effectively increased drug sensitivity and apoptosis as a consequence of reduced cell proliferation. DNMT3A-R882C/H mutations decreased apoptosis induction in part by increasing the antioxidant capacity of the cell owing to upregulation of PRDX2. Molecularly, both DNMT3A-WT and R882H/C mutants interacted with PRDX2; and R882C/H mutation-induced hypomethylation increased PRDX2 expression which enhanced cell proliferation and growth with impairment of apoptosis, thereby contributing to leukemogenesis.

The clinical and prognostic relevance of driver mutations in 203 Taiwanese patients with primary myelofibrosis

Aims We investigated the clinical and prognostic relevance of the mutational status of driver genes with allele burden and endogenous erythroid colony (EEC) growth in 203 Taiwanese patients with primary myelofibrosis (PMF). Methods Pyrosequencing was used to detect JAK2 V617F mutational status and measure allele burden, while MPL (exon 10) mutations were analysed by PCR assay and then by direct sequencing. CALR exon 9 mutations were first screened for length changes by GeneScan followed by sequencing. The allele burden of the mutated CALR gene was measured by pyrosequencing. The EEC assay was conducted using a serum-free culture system. Results The frequencies of the three driver mutations and triple-negative status were similarly distributed between pre-PMF and overt PMF patients, except that pre-PMF patients had a higher incidence of CALR type 2/type-2 like mutations and a lower JAK2 V617F allele burden. EEC growth and CALR mutations conferred favourable overall survival (OS). A lower JAK2 V617F allele burden and grade 3 bone marrow fibrosis were associated with shorter OS and decreased leukaemia-free survival (LFS). Type 2/type 2-like CAL mutations were associated with better LFS compared with type1/type 1-like mutations. Patients with triple-negative mutation status had significantly worse OS and LFS. The allele burden of CALR mutations remained unchanged, while some JAK2 V617F mutations showed clonal expansion in patients during secondary acute myeloid leukaemia transformation. Conclusions Our study showed that EEC growth, a higher JAK2 V617F allele burden and CALR mutations, especially type 2, were independent predictors for better outcomes in PMF. The allele burden of CALR mutations remained stable, but the allele burden of JAK2 V617Fmutations was variable during leukaemia transformation.