Rosalina K. L. Ip

Platelet factor 4 induces cell apoptosis by inhibition of STAT3 via up-regulation of SOCS3 expression in multiple myeloma

Platelet factor 4 (PF4) is an angiostatic chemokine that suppresses tumor growth and metastasis. We previously revealed frequent transcriptional silencing of PF4 in multiple myeloma, but the functional roles of this chemokine are still unknown. We studied the apoptotic effects of PF4 on myeloma cell lines and primary myeloma in vitro, and investigated the involved signaling pathway. The in vivo effects were also studied using a mouse model. PF4 not only suppressed myeloma-associated angiogenesis, but also inhibited growth and induced apoptosis in myeloma cells. We found that PF4 negatively regulated STAT3 and concordantly inhibited constitutive and interleukin-6-induced phosphorylation of STAT3, and down-regulated the expression of STAT3 target genes (Mcl-1, survivin and VEGF). Overexpression of constitutively activated STAT3 could rescue PF4-induced apoptotic effects. Furthermore, we found that PF4 induced the expression of SOCS3, a STAT3 inhibitor, and gene silencing of SOCS3 abolished its ability to inhibit STAT3 activation, suggesting a critical role of SOCS3 in PF4-induced STAT3 inhibition. Knockdown of LRP1, a putative PF4 receptor, could also abolish PF4-induced apoptosis and STAT3 inhibition. Finally, the tumor growth inhibitory effect of PF4 was confirmed by in vivo mouse models. Immunostaining of rabbit bone xenografts from PF4-treated mice showed induction of apoptosis of myeloma cells and inhibition of angiogenesis, which was associated with suppression of STAT3 activity. Together, our preclinical data indicate that PF4 may be a potential new targeting agent for the treatment of myeloma.

Secreted-frizzled related protein 1 is a transcriptional repression target of the t(8;21) fusion protein in acute myeloid leukemia

Secreted-frizzled related proteins (SFRPs) are modulators of the Wnt signaling pathway that is closely involved in normal and malignant hematopoiesis. Epigenetic deregulation of Wnt modulators leading to aberrant signaling has been reported in adult patients with acute myeloid leukemia (AML), but its occurrence in childhood patients with AML and the role of individual modulators are unclear. In this study, we examined SFRP1, SFRP2, SFRP4, and SFRP5 promoter methylation in 83 patients with AML (59 children and 24 adults) and found preferential SFRP1 methylation and mRNA down-regulation in the prognostically favorable subgroup of AML with t(8;21) translocation. Among the 4 genes, SFRP1 methylation independently predicted prolonged event-free and relapse-free survivals in childhood patients with nonacute promyelocytic leukemia with nonadverse cytogenetics. Mechanistically, we further demonstrated that RUNX1-ETO, the t(8;21) fusion product, specifically bound the SFRP1 promoter and repressed its transcription via a consensus RUNX binding site. In t(8;21)-leukemia cells, SFRP1 selectively inhibited canonical Wnt signaling and cellular proliferation that were associated with concomitant down-regulation of Wnt/β-catenin target genes, including CCND1 and MYC. Taken together, we identified SFRP1 as a transcriptional repression target of the t(8;21) fusion protein and demonstrated a novel mechanism of Wnt activation in a specific subtype of AML.

Minimal Residual Disease-Based Risk Stratification in Chinese Childhood Acute Lymphoblastic Leukemia by Flow Cytometry and Plasma DNA Quantitative Polymerase Chain Reaction

Minimal residual disease, or MRD, is an important prognostic indicator in childhood acute lymphoblastic leukemia. In ALL-IC-BFM 2002 study, we employed a standardized method of flow cytometry MRD monitoring for multiple centers internationally using uniformed gating, and determined the relevant MRD-based risk stratification strategies in our local patient cohort. We also evaluated a novel method of PCR MRD quantitation using peripheral blood plasma. For the bone marrow flow MRD study, patients could be stratified into 3 risk groups according to MRD level using a single time-point at day-15 (Model I) (I-A: <0.1%, I-B: 0.1–10%, I-C: >10%), or using two time-points at day-15 and day-33 (Model II) (II-A: day-15<10% and day-33<0.01%, II-B: day-15≥10% or day-33≥0.01% but not both, II-C: day-15≥10% and day-33≥0.01%), which showed significantly superior prediction of relapse (p = .00047 and <0.0001 respectively). Importantly, patients with good outcome (frequency: 56.0%, event-free survival: 90.1%) could be more accurately predicted by Model II. In peripheral blood plasma PCR MRD investigation, patients with day-15-MRD≥10−4 were at a significantly higher risk of relapse (p = 0.0117). By multivariate analysis, MRD results from both methods could independently predict patients’ prognosis, with 20–35-fold increase in risk of relapse for flow MRD I-C and II-C respectively, and 5.8-fold for patients having plasma MRD of ≥10−4. We confirmed that MRD detection by flow cytometry is useful for prognostic evaluation in our Chinese cohort of childhood ALL after treatment. Moreover, peripheral blood plasma DNA MRD can be an alternative where bone marrow specimen is unavailable and as a less invasive method, which allows close monitoring.

A polymorphism in the 3'-untranslated region of the NPM1 gene causes illegitimate regulation by microRNA-337-5p and correlates with adverse outcome in acute myeloid leukemia

Nucleophosmin, encoded by NPM1, is a haploinsufficient suppressor in hematologic malignancies. NPM1 mutations are mostly found in acute myeloid leukemia patients with normal karyotype and associated with favorable prognosis. A polymorphic nucleotide T deletion with unknown significance is present in the NPM1 3′-untranslated region. Here, we showed that the homozygous nucleotide T deletion was associated with adverse outcomes and could independently predict shortened survival in patients with de novo acute myeloid leukemia. Mechanistically, we demonstrated that the nucleotide T deletion created an illegitimate binding NPM1 for miR-337-5p, which was widely expressed in different acute myeloid leukemia subtypes and inhibited NPM1 expression. Accordingly, NPM1 levels were found to be significantly reduced and correlated with miR-337-5p levels in patients carrying a homozygous nucleotide T-deletion genotype. Together, our findings uncover a microRNA-mediated control of NPM1 expression that contributes to disease heterogeneity and suggest additional prognostic values of NPM1 in acute myeloid leukemia.

Abstract 5330: Differential expression and roles of miR-1246 and miR-1290 in multiple myeloma cancer stem cell-like subpopulation.

Multiple myeloma (MM) remains an incurable clonal plasma cell malignancy. The existence of cancer stem cell-like subpopulation in MM may explain for its unfavourable prognosis and high relapse rate. Studies have shown that CD138neg MM cells were clonogenic and could be serially transplanted, while CD138+ cells could not. We characterized CD138neg cells by checking the presence of such subpopulation and their tumorigenic properties. In concordance with literature, CD138neg cells were present in MM cell lines, and possessed higher clonogenic potential than its non-tumorigenic counterpart as shown in colony formation assay. They were more quiescent, with less than 1% in G2/M phase compared to 5-10% in CD138+ cells. They also demonstrated higher resistance to chemotherapeutic agents, including thalidomide and bortezomib. Since studies in solid tumors have demonstrated that microRNA (miRNA) plays a role in self-renewal, tumorigenicity and chemoresistance in cancer stem cells, we hypothesized that specific miRNAs are the key regulators of the cancer stem cell-like functions in MM clonogenic cells. To search for these candidates, we first identified miRNAs that are differentially expressed among the two subpopulations, CD138+ and CD138neg cells, which were isolated from MM cell lines (NCI-H929, U266 and MM17, our in-house MM cell line) using MACS-immunomagnetic separation system and subjected to Exiqon miRCURY LNA miRNA microarray analysis. Ten candidate miRNAs were identified and selected for further validation by Taqman miRNA assay. By further independent investigation using fifteen MM patient samples, concordantly we found frequent up-regulation of miR-483-5p, miR-1246, miR-1275, miR-1290 and miR-3196 in CD138neg compared to CD138+ cells. MiR-1246 (93.4%, p = 0.006) and miR-1290 (93.4%, p = 0.0015) were chosen for further examination of the potential roles in MM pathogenesis and prognosis because they showed significantly higher expression levels in CD138neg cells than the CD138+ counterparts. Preliminary results showed that inhibiting miR-1246 (p = 0.0478) and miR-1290 (p = 0.0386) reduced cell proliferation in CD138neg cells at 24h or 48h of transfection in unsorted U266 cells, while CD138+ cells were not affected. As predicted using computational algorithms (miRANDA and TargetScan), the downstream targets of the two miRNAs include SEMA6A, STK17A, PHLDA1 and KLF9, which bear functions related to MM pathogenesis or known to be tumor suppressors. Moreover, we also confirmed that their mRNA expressions were down-regulated in U266 CD138neg cells compared with CD138+ cells.Taken together, we suggest that miR-1246 and miR-1290 may play a regulatory role in MM clonogenic cell proliferation. Our findings may provide a novel insight for the microRNA-based therapeutics that specifically targets the clonogenic progenitors, which may prove to be more effective for cure of MM. Citation Format: Coty HY Cheung, Suk-Hang Cheng, Libby Li, Natalie Pui Ha Chan, Rosalina KL Ip, Chi-Keung Cheng, Margaret H.L. Ng. Differential expression and roles of miR-1246 and miR-1290 in multiple myeloma cancer stem cell-like subpopulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5330. doi:10.1158/1538-7445.AM2013-5330

Distinctive regional-specific PROS1 mutation spectrum in Southern China

Protein S (PS) is a vitamin-K dependent plasma anticoagulant important for the regulation of blood coagulation. It acts as a nonenzymatic cofactor for activated protein C (APC) in the degradation of coagulation factors Va and VIIIa [1]. Inherited PS deficiency is a prevalent genetic risk factor for venous thromboembolism in Asians affecting up to 18% of the cases [2]. However, the diagnosis of PS deficiency by current assays is difficult and complicated by multiple factors [3]. Earlier molecular studies have revealed divergent PROS1 mutation profiles among geographically and genetically close East Asians including Chinese, Japanese and Korean [4–6]. Regarding the Chinese population, Tang et al., previously reported 20 different PROS1 mutations including 15 novel ones in 21 of 40 (53%) unrelated thrombosis patients with low PS activities from Central China [4]. As geographical region-based genetic heterogeneity was evident in Han Chinese [7], it is unclear if PROS1 mutation profiles would differ even within the Chinese population. Thus, we examined the incidence and spectrum of PROS1 mutations in PS-deficient subjects in Hong Kong as Southern China, to advance our understanding on the genetic basis of PS deficiency in Chinese patients. By contrast to the findings of Tang et al., we observed a considerably higher PROS1 mutation rate (82%) and a distinctly different mutation spectrum in our cohort. Our study included 39 unrelated Chinese probands suspected of inherited PS deficiency (defined as > 2 standard deviations (SD) from the mean of gender-specific reference range, details in Supplementary Methods) referred for PROS1 genotyping. PROS1 mutations were detected by direct sequencing of all coding exons and the flanking intronic/untranslated sequences using primers as previously described [8]. For samples with no identifiable mutations, multiplex ligation-dependent probe amplification (MLPA) was performed to screen for gross PROS1 deletions/insertions (SALSA MLPA P112 PROS1 probemix, MRC-Holland, Amsterdam, The Netherlands). Of the 39 unrelated subjects, 23 were males and 16 were females (Table 1). Their median age was 52 years (range 18–72 years). Thirty-four of them (87%) had experienced thrombotic events (30 venous and 4 arterial) and the rest had family history of thrombosis. PROS1 mutations were detected in 32 of the 39 (82%) unrelated subjects and 29 of the 34 (85%) in the thrombotic group. PS activity levels were significantly lower in subjects with PROS1 mutations than subjects without the mutation (mean: − 4.8 SD vs. − 2.8 SD from mean of gender-matched reference range, P = 0.003). Overall, PROS1-mutated subjects had higher risks of venous thrombosis (84 vs. 43%, P = 0.037) than non-mutated subjects. No significant Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1123 9-018-1660-z) contains supplementary material, which is available to authorized users.

Prevalence and Clinicopathologic Significance of BRAF V600E Mutation in Chinese Multiple Myeloma Patients

Background: Previous studies in Western countries demonstrated BRAF V600E mutation only in a small subset of multiple myeloma (MM) patients. However, the prevalence and clinicopathologic significances of this mutation remain unclear in Chinese MM patients. Patients and Methods: We studied diagnostic bone marrow samples from 205 Chinese MM patients by allele‐specific PCR to detect BRAF V600E mutation and by high‐resolution melting assay to detect KRAS and NRAS mutations. The mutations were confirmed by independent assays. Results: BRAF V600E mutation was found in 9.3% of the cases, the highest prevalence hitherto reported. In addition, the mutation was significantly associated with hypercalcemia and a male predominance but not with aggressive extramedullary diseases or a high serum creatinine level as reported in Western studies. Importantly, BRAF V600E mutation was an adverse prognostic factor for overall survival in younger MM patients by subgroup analysis. Concurrent analysis of RAS mutations highlighted differential alteration spectrum of RAS signaling between Chinese and Western MM, which may suggest a unique myeloma‐related genetic profile in Chinese patients. Conclusion: Our study revealed a higher prevalence of BRAF V600E mutation in Chinese MM patients. The associated prognostic impacts on younger patients could be beneficial to risk stratification and potential application of BRAF‐targeted therapies in Chinese MM management. This is the first large‐scale study revealing the prevalence and clinicopathologic significances of BRAF V600E mutation in Chinese myeloma. Micro‐Abstract On the basis of the study of 205 Chinese patients with multiple myeloma, we demonstrated a prevalence of 9.3% of BRAF V600E mutation, the highest hitherto reported. Additionally, BRAF V600E mutation was found to be associated with hypercalcemia and a male predominance. In subgroup analysis, the BRAF V600E mutation showed prognostic impact on overall survival in younger patients (<65 years old). This is the first large‐scale study to reveal the prevalence and clinicopathologic significance of BRAF V600E mutation in myeloma in Chinese patients.

Helicase-like transcription factor is a RUNX1 target whose downregulation promotes genomic instability and correlates with complex cytogenetic features in acute myeloid leukemia

Helicase-like transcription factor is a SWI/SNF chromatin remodeling factor involved in various biological processes. However, little is known about its role in hematopoiesis. In this study, we measured helicase-like transcription factor mRNA expression in the bone marrow of 204 adult patients with de novo acute myeloid leukemia. Patients were dichotomized into low and high expression groups at the median level for clinicopathological correlations. Helicase-like transcription factor levels were dramatically reduced in the low expression patient group compared to those in the normal controls (n=40) (P<0.0001). Low helicase-like transcription factor expression correlated positively with French-American-British M4/M5 subtypes (P<0.0001) and complex cytogenetic abnormalities (P=0.02 for ≥3 abnormalities; P=0.004 for ≥5 abnormalities) but negatively with CEBPA double mutations (P=0.012). Also, low expression correlated with poorer overall (P=0.005) and event-free (P=0.006) survival in the intermediate-risk cytogenetic subgroup. Consistent with the more aggressive disease associated with low expression, helicase-like transcription factor knockdown in leukemic cells promoted proliferation and chromosomal instability that was accompanied by downregulation of mitotic regulators and impaired DNA damage response. The significance of helicase-like transcription factor in genome maintenance was further indicated by its markedly elevated expression in normal human CD34+ hematopoietic stem cells. We further demonstrated that helicase-like transcription factor was a RUNX1 target and transcriptionally repressed by RUNX1-ETO and site-specific DNA methylation through a duplicated RUNX1 binding site in its promoter. Taken together, our findings provide new mechanistic insights on genomic instability linked to helicase-like transcription factor deregulation, and strongly suggest a tumor suppressor function of the SWI/SNF protein in acute myeloid leukemia.

Abstract 1114: Downregulated miR-337-5p is associated with unfavorable features in acute myeloid leukemia and may play a role in modulating the extracellular signal-regulated kinase pathway

We previously revealed that a homozygous deletion T polymorphism in the 3’-untranslated region of the Nucleophosmin (NPM1) gene is associated with adverse outcomes and can independently predict survivals in patients with de novo acute myeloid leukemia (AML). This polymorphism creates an illegitimate binding site for miR-337-5p, which is expressed in different AML subtypes. We speculate that the delT-carrying NPM1 mRNA competes with as yet unidentified oncogenes for miR-337-5p binding, thereby activating their functions in trans and perturbing normal microRNA regulation. In this study, we aim to investigate the clinicopathological and biological significance of miR-337-5p expression in AML. Using real-time RT-PCR, we measured miR-337-5p expression in bone marrow of 156 adult patients with de novo AML excluding acute promyelocytic leukemia. Patients were dichotomized into low and high expression group at the median level for clinicopathological correlations. miR-337-5p levels were significantly reduced in the low expression patient group than the normal controls (n = 27) (P Citation Format: Rosalind Lie, Chi Keung Cheng, Natalie Pui Ha Chan, Rosalina Ip, Nelson Chan, Joyce Cheung, Margaret Ng. Downregulated miR-337-5p is associated with unfavorable features in acute myeloid leukemia and may play a role in modulating the extracellular signal-regulated kinase pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1114.

Abstract 2232: Down-regulation of dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A mediated by miR-1246 and miR-1290 in multiple myeloma cancer stem-like cells

Multiple Myeloma (MM) is a clonal plasma cell derived malignancy characterized with high relapse rate and fatality despite recent therapeutic advances. The existence of the rare population of CD138neg cancer stem-like cells had been identified and demonstrated to bear higher clonogenicity and self-renewal capacity when compared to the CD138pos major population. Besides, CD138neg MM cells are more resistant to common therapeutic agents treating MM including dexamethasone, lenalidomide and bortezomib. Therefore, targeting MM cancer-stem like cells could be critical to achieve a cure in MM. Previously, our lab had identified 2 microRNAs, miR-1246 and miR-1290 that are overexpressed in CD138neg MM cells in both cell lines and patient samples. Their target genes are being studied in the present study. CD138pos and CD138neg MM cells were isolated using MACS-immunomagnetic system and the purity of both CD138neg and CD138pos fraction were accessed by flow cytometry using PE anti-human CD138 antibody. The purity of each fraction was over 95% in 4 cell lines (U266, RPMI8226, OPM2 and KMS-12-PE) used. mRNA level of 7 putative targets (DYRK1A, KDM5A, NKRF, STK17A, PHLDA1, FOXA1, SCAI) chosen from TargetScan 6.2, miRanda or previous studies, which are related to tumor development in other cancers, of miR-1246 or miR-1290 were examined using RT-qPCR. Among the 7 putative targets, DYRK1A, KDM5A and NKRF were down-regulated in all four cell lines used. 3′UTR of the 3 down-regulated targets were cloned into pmirGLO Dual-Luciferase miRNA Target Expression Vector, which were co-transfected with miRNA-mimic into 293FT cells. Down-regulation of luciferase signal was observed in DYRK1A (p Citation Format: Terry Hei Yan Wong, Coty HY Cheung, Natalie Pui Ha Chan, Rosalina KL Ip, Chi-Keung Cheng, Kitty Ka Yan Ng, Margaret H.L. Ng. Down-regulation of dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A mediated by miR-1246 and miR-1290 in multiple myeloma cancer stem-like cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2232. doi:10.1158/1538-7445.AM2015-2232