Association between plasma copper levels and first stroke: a community-based nested case-control study.

BACKGROUND AND AIMS: Uncertainty remains regarding the association between the risk of stroke and plasma copper levels in population with copper mostly in normal range due to limited data. We examined the association between baseline plasma copper and risk of first stroke in Chinese community-dwelling population. METHODS: We conducted a nested case control study from 'H-type Hypertension and Stroke Prevention and Control Project'. A total of 1255 first stroke cases and 1255 controls matched for age, sex and study site were included in the analysis. Conditional logistic regression analyses were performed to evaluate the association between plasma copper and first stroke. RESULTS: The overall mean of copper was 15.90 (2.66) µmol/L. In total, 94.26% participants' copper concentration was in the normal range by Mayo Clinic laboratory reference values. Smoothing curve showed that the associations of plasma copper with first stroke and its subtypes were linear. Each standard deviation (SD) increment of plasma copper was independently and positively associated with risk of first stroke [odds ratio (OR): 1.17, 95% confidence interval (CI): 1.07-1.28]. The multivariable ORs with 95% CIs for total stroke, ischemic stroke and hemorrhagic stroke in the highest versus the lowest quartile of plasma copper were 1.49 (1.16-1.90; P-trend = 0.001), 1.46 (1.12-1.92; P-trend = 0.004) and 2.05 (0.95-4.38; P-trend = 0.050), respectively. CONCLUSIONS: Baseline plasma copper was positively associated with risk of first ischemic stroke in an approximately linear fashion among Chinese community population (80.32% hypertensives), although their copper levels were mostly within the normal range according to current reference values. Our findings warrant additional investigation.

Serum folate modified the association between low-density lipoprotein cholesterol and carotid intima-media thickness in Chinese hypertensive adults.

BACKGROUND AND AIMS: While folate is known for its importance in cardiovascular health, it is unknown whether folate status can modify the association between low-density lipoprotein cholesterol (LDL-C) and carotid intima-media thickness (CIMT). We aimed to investigate this question in a Chinese hypertensive population, who are at high-risk of low folate and atherosclerosis. METHODS AND RESULTS: This report included 14,970 hypertensive adults (mean age 64.5 years; 40.3% male) from the China Stroke Primary Prevention Trial (CSPPT) and analyzed the fasting serum LDL-C and folate, and CIMT data obtained at the last follow-up visit. LDL-C was calculated using the Friedewald equation. Serum folate levels were measured by chemiluminescent immunoassay. CIMT was measured by ultrasound. Non-parametric smoothing plots, multivariate linear regression analysis, subgroup analyses and interaction testing were performed to examine the LDL-C-CIMI relationship and effect modification by folate. Consistent with graphic plots, multivariate linear regression showed that LDL-C levels were independently and positively associated with CIMT (ß = 7.69, 95%CI: 5.76-9.62). More importantly, the relationship between LDL-C and CIMT was significantly attenuated with increasing serum folate levels (1st tertile: ß = 10.06, 95%CI: 6.67-13.46; 2nd tertile: ß = 6.81, 95%CI: 3.55-10.07; 3rd tertile: ß = 5.96, 95%CI: 2.55-9.36; P-interaction = 0.045). Subgroup analyses showed the association between LDL-C and CIMT across serum folate tertiles was robust among various strata (all P-interaction >0.05). CONCLUSIONS: Among Chinese hypertensive adults, the serum folate levels could modify the association between LDL-C and CIMT. Our findings, if further confirmed, have important clinical implications.

Plasma Magnesium Concentrations and Risk of Incident Cancer in Adults with Hypertension: A Nested Case-Control Study.

OBJECTIVE: The association between plasma magnesium and risk of incident cancer remains inconclusive in previous studies. We aimed to investigate the prospective relationship of baseline plasma magnesium concentrations with the risk of incident cancer and to examine possible effect modifiers. METHODS: A nested case-control study with 228 incident cancer cases and 228 matched controls was conducted using data from the China Stroke Primary Prevention Trial (CSPPT), a randomized, double-blind, controlled trial, conducted from May 2008 to August 2013. Study outcomes included incident cancer and its subtypes. RESULTS: When plasma magnesium concentrations were assessed as quartiles, a significantly higher incident risk of total cancer was found in participants in quartile 1 (<0.76 mmol/L; odds ratio [OR] = 2.70; 95% CI: 1.33-5.49) and quartile 4 (≥0.89 mmol/L; OR = 2.05; 95% CI: 1.12-3.76), compared with those in quartile 3 (0.83 to <0.89 mmol/L). In cancer site-specific analyses, similar trends were found for gastrointestinal cancer, esophageal cancer, gastric cancer, breast cancer, lung cancer, and other cancers. Furthermore, none of the variables, including age, sex, current smoking status, current alcohol intake, BMI, systolic blood pressure, and total cholesterol levels at baseline significantly modified the association between plasma magnesium and cancer risk. CONCLUSIONS: Both low and high plasma magnesium concentrations were significantly associated with an increased incident risk of cancer, compared with the reference concentrations of 0.83 to <0.89 mmol/L among hypertensive adults.

EZH2 phosphorylation by JAK3 mediates a switch to noncanonical function in natural killer/T-cell lymphoma.

The best-understood mechanism by which EZH2 exerts its oncogenic function is through polycomb repressive complex 2 (PRC2)-mediated gene repression, which requires its histone methyltransferase activity. However, small-molecule inhibitors of EZH2 that selectively target its enzymatic activity turn out to be potent only for lymphoma cells with EZH2-activating mutation. Intriguingly, recent discoveries, including ours, have placed EZH2 into the category of transcriptional coactivators and thus raised the possibility of noncanonical signaling pathways. However, it remains unclear how EZH2 switches to this catalytic independent function. In the current study, using natural killer/T-cell lymphoma (NKTL) as a disease model, we found that phosphorylation of EZH2 by JAK3 promotes the dissociation of the PRC2 complex leading to decreased global H3K27me3 levels, while it switches EZH2 to a transcriptional activator, conferring higher proliferative capacity of the affected cells. Gene expression data analysis also suggests that the noncanonical function of EZH2 as a transcriptional activator upregulates a set of genes involved in DNA replication, cell cycle, biosynthesis, stemness, and invasiveness. Consistently, JAK3 inhibitor was able to significantly reduce the growth of NKTL cells, in an EZH2 phosphorylation-dependent manner, whereas various compounds recently developed to inhibit EZH2 methyltransferase activity have no such effect. Thus, pharmacological inhibition of JAK3 activity may provide a promising treatment option for NKTL through the novel mechanism of suppressing noncanonical EZH2 activity.

EZH2 overexpression in natural killer/T-cell lymphoma confers growth advantage independently of histone methyltransferase activity.

The role of enhancer of zeste homolog 2 (EZH2) in cancer is complex and may vary depending on the cellular context. We found that EZH2 is aberrantly overexpressed in the majority of natural killer/T-cell lymphoma (NKTL), an aggressive lymphoid malignancy with very poor prognosis. We show that EZH2 upregulation is mediated by MYC-induced repression of its regulatory micro RNAs and EZH2 exerts oncogenic properties in NKTL. Ectopic expression of EZH2 in both primary NK cells and NKTL cell lines leads to a significant growth advantage. Conversely, knock-down of EZH2 in NKTL cell lines results in cell growth inhibition. Intriguingly, ectopic EZH2 mutant deficient for histone methyltransferase activity is also able to confer growth advantage and rescue growth inhibition on endogenous EZH2 depletion in NKTL cells, indicating an oncogenic role of EZH2 independent of its gene-silencing activity. Mechanistically, we show that EZH2 directly promotes the transcription of cyclin D1 and this effect is independent of its enzymatic activity. Furthermore, depletion of EZH2 using a PRC2 inhibitor 3-deazaneplanocin A significantly inhibits growth of NK tumor cells. Therefore, our study uncovers an oncogenic role of EZH2 independent of its methyltransferase activity in NKTL and suggests that targeting EZH2 may have therapeutic usefulness in this lymphoma.

Deregulated MIR335 that targets MAPK1 is implicated in poor outcome of paediatric acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) is the most common paediatric malignancy. Although 90% of patients are now long-term survivors, the remaining 10% have poor outcome predominantly due to drug resistance. In this study, we carried out genome-wide microRNA (miRNA) microarray analysis on diagnostic bone marrow samples to determine miRNA expression profiles associated with poor outcome in ALL. A reduced expression of MIR335 was identified as the most significant miRNA abnormality associated with poor outcome. It is well known that glucocorticoid (GC) resistance is one of the major reasons contributing to poor outcome. We show that exogenous expression of MIR335 in ALL cells increases sensitization to prednisolone-mediated apoptosis. Moreover, we demonstrate that MAPK1 is a novel target of MIR335, and that MEK/ERK inhibitor treatment enhanced prednisolone-induced cell death through the activation of BIM (BCL2L11). These results provide a possible underlying molecular mechanism to explain the association between reduced MIR335 with poor clinical outcome, and suggest that approaches to re-introduce MIR335 expression or override MAPK1 activity may offer promising therapeutic strategies in the treatment of ALL.

The histone methyltransferase inhibitor, DZNep, up-regulates TXNIP, increases ROS production, and targets leukemia cells in AML.

Recent studies have shown that 3-Deazaneplanocin A (DZNep), a histone methyltransferase inhibitor, disrupts polycomb-repressive complex 2 (PRC2), and preferentially induces apoptosis in cancer cells, including acute myeloid leukemia (AML). However, the underlying molecular mechanisms are not well understood. The present study demonstrates that DZNep induces robust apoptosis in AML cell lines, primary cells, and targets CD34(+)CD38(-) leukemia stem cell (LSC)-enriched subpopulations. Using RNA interference (RNAi), gene expression profiling, and ChIP, we identified that TXNIP, a major redox control molecule, plays a crucial role in DZNep-induced apoptosis. We show that disruption of PRC2, either by DZNep treatment or EZH2 knockdown, reactivates TXNIP, inhibits thioredoxin activity, and increases reactive oxygen species (ROS), leading to apoptosis. Furthermore, we show that TXNIP is down-regulated in AML and is a direct target of PRC2-mediated gene silencing. Consistent with the ROS accumulation on DZNep treatment, we also see a signature of endoplasmic reticulum (ER) stress-regulated genes, commonly associated with cell survival, down-regulated by DZNep. Taken together, we uncover a novel molecular mechanism of DZNep-mediated apoptosis and propose that EZH2 may be a potential new target for epigenetic treatment in AML.

Dysregulated microRNAs affect pathways and targets of biologic relevance in nasal-type natural killer/T-cell lymphoma.

We performed a comprehensive genome-wide miRNA expression profiling of extranodal nasal-type natural killer/T-cell lymphoma (NKTL) using formalin-fixed paraffin-embedded tissue (n = 30) and NK cell lines (n = 6) compared with normal NK cells, with the objective of understanding the pathogenetic role of miRNA deregulation in NKTL. Compared with normal NK cells, differentially expressed miRNAs in NKTL are predominantly down-regulated. Re-expression of down-regulated miRNAs, such as miR-101, miR-26a, miR26b, miR-28-5, and miR-363, reduced the growth of the NK cell line and modulated the expression of their predicted target genes, suggesting the potential functional role of the deregulated miRNAs in the oncogenesis of NKTL. Taken together, the predicted targets whose expression is inversely correlated with the expression of deregulated miRNA in NKTL are significantly enriched for genes involved in cell cycle-related, p53, and MAPK signaling pathways. We also performed immunohistochemical validation for selected target proteins and found overexpression of MUM1, BLIMP1, and STMN1 in NKTL, and notably, a corresponding increase in MYC expression. Because MYC is known to cause repression of miRNA expression, it is possible that MYC activation in NKTL may contribute to the suppression of the miRNAs regulating MUM1, BLIMP1, and STMN1.

The pro-metastasis tyrosine phosphatase, PRL-3 (PTP4A3), is a novel mediator of oncogenic function of BCR-ABL in human chronic myeloid leukemia.

BACKGROUND: Resistance to tyrosine kinase inhibitors (TKIs) remains a challenge in management of patients with chronic myeloid leukemia (CML). A better understanding of the BCR-ABL signalling network may lead to better therapy. FINDINGS: Here we report the discovery of a novel downstream target of BCR-ABL signalling, PRL-3 (PTP4A3), an oncogenic tyrosine phosphatase. Analysis of CML cancer cell lines and CML patient samples reveals the upregulation of PRL-3. Inhibition of BCR-ABL signalling either by Imatinib or by RNAi silencing BCR-ABL reduces PRL-3 and increases cleavage of PARP. In contrast, the amount of PRL-3 protein remains constant or even increased in response to Imatinib treatment in drug resistant cells expressing P210 T315I. Finally, analysis with specific shRNA shows PRL-3 involvement in the proliferation and self-renewal of CML cells. CONCLUSIONS: These data support a role for PRL-3 in BCR-ABL signalling and CML biology and may be a potential therapeutic target downstream of BCR-ABL in TKI resistant mutant cells.

Association between serum calcium levels and first stroke: A community-based nested case-control study.

Background: Evidence from epidemiologic studies has been limited and inconsistent regarding the role of serum calcium in stroke incidence risk. We aimed to evaluate the association between serum albumin-corrected calcium and the risk of the first stroke in the Chinese community-dwelling population. Methods: The study sample population was drawn from the "H-type Hypertension and Stroke Prevention and Control Project." Using a nested case-control study, a total of 1,255 first-stroke cases and 1,255 controls matched for age, sex, and village were included in the final data analysis. We measured the serum calcium by inductively coupled plasma mass spectrometry and assessed the associations between serum albumin-corrected calcium and first stroke using conditional logistic regression. Results: The overall mean (SD) serum albumin-corrected calcium was 8.9 (0.6) mg/dl. Compared with the middle tertile (8.7-9.1 mg/dl), the multivariate-adjusted odds ratios (95% CIs) of first total stroke associated with the lowest tertile and the highest tertile of serum albumin-corrected calcium were 1.37 (1.10, 1.70) and 1.30 (1.04, 1.62), respectively. Similar trends were observed for the first ischemic stroke. Consistently, restricted cubic spline showed a U-shaped association between serum albumin-corrected calcium and risk of total stroke and ischemic stroke. However, serum albumin-corrected calcium had no significant effect on first hemorrhagic stroke. No significant effect modification was observed in the subgroup analysis. Conclusions: Our results suggested a U-shaped association between serum calcium and first stroke; both low and high serum calcium levels were associated with an increased risk of the first stroke in the Chinese population.

Association between baseline brachial-ankle pulse wave velocity and short-term risk of first stroke among Chinese hypertensive adults.

Little information is available on the association between brachial-ankle pulse wave velocity (baPWV) and the risk of stroke in Chinese H-type hypertension patients. Therefore, our study aimed to assess this association between baseline baPWV and short-term risk of first stroke and to propose a cutoff value of baPWV that could predict near cerebrovascular events. A total of 9787 hypertension patients without preexisting stroke who underwent baPWV measurement were included. The primary end points were first symptomatic stroke. Secondary end points were first ischemic stroke and first hemorrhagic stroke. During a median follow-up of 20.8 months, there was a total of 138 first strokes including 123 first ischemic strokes and 15 first hemorrhagic strokes. When baPWV was categorized in quartiles, the higher risks of first stroke (HR = 1.52; 95% CI: 1.05-2.21) and first ischemic stroke (HR = 1.53; 95% CI: 1.03-2.26) were found in participants in quartile 4 (≥21.31 m/s), compared with those in quartile 1-3 (<21.31 m/s). In receiver operating characteristic curve analysis, the best cutoff value of baPWV that could predict first stroke was 21.43 m/s. Higher baPWV (≥21.43 m/s) was significantly associated with increased risk of first stroke (HR = 1.60; 95% CI: 1.10-2.32) and first ischemic stroke (HR = 1.60; 95% CI: 1.08-2.37). In conclusion, higher baPWV levels were associated with an increased risk of first stroke among Chinese H-type hypertensive patients. In addition, a cutoff value of 21.43 m/s of baPWV was proposed that could predict the next two years' cerebrovascular events.

Plasma 25-hydroxyvitamin D3 concentrations and incident risk of ischemic stroke in rural Chinese adults: New insight on ceiling effect.

OBJECTIVE: The aim of this study was to investigate the relationship between 25-hydroxyvitamin D3 (25[OH]D3) and ischemic stroke and its potential modifying factors in rural Chinese adults. METHODS: This nested case-control study was drawn from the H-type Hypertension and Stroke Prevention and Control Project, a community-based, prospective, observational study. Plasma 25(OH)D3 was measured by liquid chromatography with tandem quadrupole mass spectrometry. All stroke records came from the Chinese Center for Disease Control and Prevention. Multiple logistic regression models were used to evaluate the association between 25(OH)D3 and risk of ischemic stroke. RESULTS: We included 1079 participants with ischemic stroke and 1079 matched controls. Due to a non-linear relationship, the analyses were stratified by 25(OH)D3. For those with 25(OH)D3 < 20 ng/mL, there was a 15% reduction in the risk of ischemic stroke for each SD increment in 25(OH)D3 (odds ratio, 0.85; 95% confidence interval, 0.73-0.99). Compared with the lowest-tertile group, the risk of ischemic stroke decreased by 39% (odds ratio, 0.61; 95% confidence interval, 0.41-0.89) in the highest-tertile group. Furthermore, two effect modifiers were identified: diabetes and homocysteine level. Although participants with 25(OH)D3 ≥ 20 ng/mL had the lowest risk of ischemic stroke overall, there was no dose-response association within that range. CONCLUSIONS: An inverse dose-response association between 25(OH)D3 and incident risk of ischemic stroke in rural Chinese adults was only observed in those with 25(OH)D3 < 20 ng/mL, along with two effect modifiers. Higher levels of 25(OH)D3 did not confer additional benefit.

Enhanced activation of STAT pathways and overexpression of survivin confer resistance to FLT3 inhibitors and could be therapeutic targets in AML.

To further investigate potential mechanisms of resistance to FLT3 inhibitors, we developed a resistant cell line by long-term culture of MV4-11 cells with ABT-869, designated as MV4-11-R. Gene profiling reveals up-regulation of FLT3LG (FLT3 ligand) and BIRC5 (survivin), but down-regulation of SOCS1, SOCS2, and SOCS3 in MV4-11-R cells. Hypermethylation of these SOCS genes leads to their transcriptional silencing. Survivin is directly regulated by STAT3. Stimulation of the parental MV4-11 cells with FLT3 ligand increases the expression of survivin and phosphorylated protein STAT1, STAT3, STAT5. Targeting survivin by short-hairpin RNA (shRNA) in MV4-11-R cells induces apoptosis and augments ABT-869-mediated cytotoxicity. Overexpression of survivin protects MV4-11 from apoptosis. Subtoxic dose of indirubin derivative (IDR) E804 resensitizes MV4-11-R to ABT-869 treatment by inhibiting STAT signaling activity and abolishing survivin expression. Combining IDR E804 with ABT-869 shows potent in vivo efficacy in the MV4-11-R xenograft model. Taken together, these results demonstrate that enhanced activation of STAT pathways and overexpression of survivin are important mechanisms of resistance to ABT-869, suggesting that the STAT pathways and survivin could be potential targets for reducing resistance developed in patients receiving FLT3 inhibitors.

High-Density Lipoprotein Cholesterol and the Risk of First Ischemic Stroke in a Chinese Hypertensive Population.

BACKGROUND AND PURPOSE: Elevated high-density lipoprotein cholesterol (HDL-C) levels have displayed protection against cardiovascular disease. However, the association between specific lipoprotein classes and first ischemic stroke (IS) has not been well defined, particularly in higher-risk hypertensive populations. Our study evaluated the associations of HDL-C with first IS in a Chinese hypertensive population. METHODS: The study population was obtained from a community-based cohort study of hypertension in Lianyungang and Rongcheng, China. A nested case-control design was used that included 2463 identified first IS cases and 2463 controls matched by age ± 1 year, sex, and region. RESULTS: After adjusting for potential confounders, HDL-C was inversely associated with first IS (adjusted odds ratio [aOR]: 0.91; 95% confidence interval [CI]: 0.85-0.98). HDL-C levels of at least 65.4 mg/dL displayed a significant protective effect for first IS (aOR: 0.82; 95% CI: 0.69-0.98). Conversely, adverse effects of first IS were observed for low-density lipoprotein cholesterol (LDL-C) levels ≥138.1 mg/dL (aOR: 1.20; 95% CI: 1.02-1.42) and triglyceride (TG) levels ≥140.8 mg/dL (aOR: 1.27; 95% CI: 1.09-1.49). The risk associations of LDL-C and TG with first IS were attenuated in the presence of high HDL-C (≥53.0 mg/dL); an increased risk of first IS was only found in the presence of low HDL-C (<53.0 mg/dL) when LDL-C (aOR: 1.66; 95% CI: 1.19-2.31) and TG (aOR: 1.47; 95% CI: 1.17-1.84) were combined with HDL-C for analysis. CONCLUSION: In this community-based Chinese hypertensive population, higher HDL-C was a significant protective factor of first IS. These data add to the evidence describing the relationship between lipids and IS and suggest that HDL-C maybe is a marker of IS risk in Chinses hypertensive population.

Sex difference in the association between plasma selenium and first stroke: a community-based nested case-control study.

BACKGROUND: To date, there is no clearly defined association between plasma selenium levels and first stroke. We aimed to investigate the association between baseline plasma selenium and first stroke risk in a community-based Chinese population. METHODS: Using a nested case-control study design, a total of 1255 first stroke cases and 1255 matched controls were analyzed. Participant plasma selenium concentrations were measured by inductively coupled plasma mass spectrometry (ICP-MS), and the association of plasma selenium with first stroke risk was estimated by conditional logistic regression models. RESULTS: Overall, a non-linear negative association between plasma selenium and first total stroke and first ischemic stroke risks was found in males but not in females. Compared with participants with lower selenium levels (tertile 1-2, < 94.1 ng/mL), participants with higher selenium levels (tertile 3, ≥ 94.1 ng/mL) had significantly lower risks of first total stroke (OR 0.63; 95% CI 0.48, 0.83) and first ischemic stroke (OR 0.61; 95% CI 0.45, 0.83) in males but not in females with first total stroke (OR 0.92; 95% CI 0.69, 1.22) and first ischemic stroke (OR 0.89; 95% CI 0.65, 1.22). Furthermore, a stronger association between plasma selenium and first total stroke was found in males with higher vitamin E levels (≥ 13.5 µg/mL vs. < 13.5 µg/mL P-interaction = 0.007). No significant association was observed between plasma selenium and first hemorrhagic stroke risk in either males or females. CONCLUSION: Our study indicated a significant, non-linear, negative association between plasma selenium and first stroke in males but not in females. TRIAL REGISTRATION: ChiCTR1800017274 .

Achieving blood pressure control targets in hypertensive patients of rural China - a pilot randomized trial.

BACKGROUND: This study aimed to test the feasibility and titration methods used to achieve specific blood pressure (BP) control targets in hypertensive patients of rural China. METHODS: A randomized, controlled, open-label trial was conducted in Rongcheng, China. We enrolled 105 hypertensive participants aged over 60 years, and who had no history of stroke or cardiovascular disease. The patients were randomly assigned to one of three systolic-BP target groups: standard: 140 to < 150 mmHg; moderately intensive: 130 to < 140 mmHg; and intensive: < 130 mmHg. The patients were followed for 6 months. DISCUSSION: The optimal target for systolic blood pressure (SBP) lowering is still uncertain worldwide and such information is critically needed, especially in China. However, in China the rates of awareness, treatment and control are only 46.9%, 40.7%, and 15.3%, respectively. It is challenging to achieve BP control in the real world and it is very important to develop population-specific BP-control protocols that fully consider the population's characteristics, such as age, sex, socio-economic status, compliance with medication, education level, and lifestyle. This randomized trial showed the feasibility and safety of the titration protocol to achieve desirable SBP targets (< 150, < 140, and < 130 mmHg) in a sample of rural, Chinese hypertensive patients. The three BP target groups had similar baseline characteristics. After 6 months of treatment, the mean SBP measured at an office visit was 137.2 mmHg, 131.1 mmHg, and 124.2 mmHg, respectively, in the three groups. Home BP and central aortic BP measurements were also obtained. At 6 months, home BP measurements (2 h after drug administration) showed a mean SBP of 130.9 mmHg in the standard group, 124.9 mmHg in the moderately intensive group, and 119.7 mmHg in the intensive group. No serious adverse events were recorded over the 6-month study period. Rates of adverse events, including dry cough, palpitations, and arthralgia, were low and showed no significant differences between the three groups. This trial provided real-world experience and laid the foundation for a future, large-scale, BP target study. TRIAL REGISTRATION: Feasibility Study of the Intensive Systolic Blood Pressure Control; ClinicalTrials.gov, ID: NCT02817503. Registered retrospectively on 29 June 2016.

In vivo activity of ABT-869, a multi-target kinase inhibitor, against acute myeloid leukemia with wild-type FLT3 receptor.

Neoangiogenesis plays an important role in leukemogenesis. We investigated the in vivo anti-leukemic effect of ABT-869 against AML with wild-type FLT3 using RFP transfected HL60 cells with in vivo imaging technology on both the subcutaneous and systemic leukemia xenograft models. ABT-869 showed a five-fold inhibition of tumor growth in comparison with vehicle control. IHC analysis revealed that ABT-869 decreased p-VEGFR1, Ki-67 labeling index, VEGF and remarkably increased apoptotic cells in the xenograft models. ABT-869 also reduced the leukemia burden and prolonged survival. Our study supports the rationale for clinically testing an anti-angiogenesis agent in AML with wild-type FLT3.

Belinostat exerts antitumor cytotoxicity through the ubiquitin-proteasome pathway in lung squamous cell carcinoma.

There have been advances in personalized therapy directed by molecular profiles in lung adenocarcinoma, but not in lung squamous cell carcinoma (SCC). The lack of actionable driver oncogenes in SCC has restricted the use of small-molecule inhibitors. Here, we show that SCC cell lines displayed differential sensitivities to belinostat, a pan-histone deacetylase inhibitor. Phosphoproteomic analysis of belinostat-treated SCC cells revealed significant downregulation of the MAPK pathway, along with the induction of apoptosis. In cisplatin-resistant cells that demonstrated aberrant MAPK activation, combined treatment with belinostat significantly inhibited cisplatin-induced ERK phosphorylation and exhibited strong synergistic cytotoxicity. Furthermore, belinostat transcriptionally upregulated the F-box proteins FBXO3 and FBXW10, which directly targeted son of sevenless (SOS), an upstream regulator of the MAPK pathway, for proteasome-mediated degradation. Supporting this, suppression of SOS/ERK pathway by belinostat could be abrogated by inhibiting proteasomal activity either with bortezomib or with siRNA knockdown of FBXO3/FBXW10. Taken together, these preclinical data offer a novel understanding of the epigenetic mechanism by which belinostat exerts its cytotoxicity and supports the combination with cisplatin in clinical settings for chemorefractory SCC tumors.

VS-5584 mediates potent anti-myeloma activity via the upregulation of a class II tumor suppressor gene, RARRES3 and the activation of Bim.

The PI3K/mTOR/AKT pathway is an integral regulator of survival and drug resistance in multiple myeloma (MM). VS-5584 was synthesized with dual-specific and equipotent activity against mTORC1/2 and all four Class I PI3K isoforms so as to durably inhibit this pathway. We show that VS-5584 is highly efficacious against MM cell lines even in the presence of IL-6 and IGF-1 and that this growth inhibition is partially dependent on Bim. Importantly, VS-5584 triggers apoptosis in patient cells with a favorable therapeutic index. Gene expression profiling revealed a VS-5584-induced upregulation of RARRES3, a class II tumor suppressor gene. MM patient databases, UAMS and APEX, show that RARRES3 is under-expressed in 11q13 subsets which correlates with the reduced effectiveness of VS-5584 in 11q13 cell lines. Silencing RARRES3 expression significantly rescues VS-5584-induced cell death and increases cyclin D2 expression but not cyclin D1 or other cyclins implying a role for RARRES3 in cell cycle arrest. In vivo, VS-5584 significantly reduces the tumor burden of MM mouse xenografts. We further identified that VS-5584 synergised with Dexamethasone, Velcade, and exceptionally so with HDAC inhibitor, Panobinostat. Interestingly, this was consistently observed in several patient samples, proposing a promising novel clinical strategy for combination treatment especially in relapsed/refractory patients.

Inhibition of LIN28B impairs leukemia cell growth and metabolism in acute myeloid leukemia.

BACKGROUND: Current conventional chemotherapy for acute myeloid leukemia (AML) can achieve remission in over 70% of patients, but a majority of them will relapse within 5 years despite continued treatment. The relapse is postulated to be due to leukemia stem cells (LSCs), which are different from normal hematopoietic stem cells (HSCs). LIN28B is microRNA regulator and stem cell reprogramming factor. Overexpression of LIN28B has been associated with advance human malignancies and cancer stem cells (CSCs), including AML. However, the molecular mechanism by which LIN28B contributes to the development of AML remains largely elusive. METHODS: We modulated LIN28B expression in AML and non-leukemic cells and investigated functional consequences in cell proliferation, cell cycle, and colony-forming assays. We performed a microarray-based analysis for LIN28B-silencing cells and interrogated gene expression data with different bioinformatic tools. AML mouse xenograft model was used to examine the in vivo function of LIN28B. RESULTS: We demonstrated that targeting LIN28B in AML cells resulted in cell cycle arrest, inhibition of cell proliferation and colony formation, which was induced by de-repression of let-7a miRNA. On the other hand, overexpression of LIN28B promoted cell proliferation. Data point to a mechanism where that inhibition of LIN28B induces metabolic changes in AML cells. IGF2BP1 was confirmed to be a novel downstream target of LIN28B via let-7 miRNA in AML. Notably, ectopic expression of LIN28B increased tumorigenicity, while silencing LIN28B led to slow tumor growth in vivo. CONCLUSIONS: In sum, these results uncover a novel mechanism of an important regulatory signaling, LIN28B/let-7/IGF2BP1, in leukemogenesis and provide a rationale to target this pathway as effective therapeutic strategy.