Nianxiang Zhang

Ebf1 or Pax5 haploinsufficiency synergizes with STAT5 activation to initiate acute lymphoblastic leukemia

STAT5 is abnormally activated in patients with acute lymphoblastic leukemia, and increased STAT5 activation synergizes with PAX5 and EBF1 to induce disease.

Prognostic impact and targeting of CRM1 in acute myeloid leukemia

Chromosomal region maintenance 1 (CRM1) is a nuclear export receptor recognizing proteins bearing a leucine-rich nuclear export signal. CRM1 is involved in nuclear export of tumor suppressors such as p53. We investigated the prognostic significance of CRM1 in acute myeloid leukemia (AML) and effects of a novel small-molecule selective inhibitor of CRM1. CRM1 protein expression was determined in 511 newly diagnosed AML patients and was correlated with mouse double minute 2 (MDM2) and p53 levels. High CRM1 expression was associated with short survival of patients and remained an adverse prognostic factor in multivariate analysis. CRM1 inhibitor KPT-185 induced mainly full-length p53 and apoptosis in a p53-dependent manner, whereas inhibition of proliferation was p53 independent. Patient samples with p53 mutations showed low sensitivity to KPT-185. Nuclear retention of p53 induced by CRM1 inhibition synergized with increased levels of p53 induced by MDM2 inhibition in apoptosis induction. KPT-185 and Nutlin-3a, alone and in combination, induced synergistic apoptosis in patient-derived CD34(+)/CD38(-) AML, but not in normal progenitor cells. Data suggest that CRM1 exerts an antiapoptotic function and is highly prognostic in AML. We propose a novel combinatorial approach for the therapy of AML, aimed at maximal activation of p53-mediated apoptosis by concomitant MDM2 and CRM1 inhibition.

Highly Phosphorylated FOXO3A Is an Adverse Prognostic Factor in Acute Myeloid Leukemia

Purpose: The Forkhead transcription factors (FOXO) are tumor suppressor genes regulating differentiation, metabolism, and apoptosis that functionally interact with signal transduction pathways shown to be deregulated and prognostic in acute myelogenous leukemia (AML). This study evaluated the level of expression and the prognostic relevance of total and phosphorylated FOXO3A protein in AML. Experimental Design: We used reverse-phase protein array methods to measure the level of total and phosphoprotein expression of FOXO3A, in leukemia-enriched protein samples from 511 newly diagnosed AML patients. Results: The expression range was similar to normal CD34+ cells and similar in blood and marrow. Levels of total FOXO3A were higher at relapse compared with diagnosis. Levels of pFOXO3A or the ratio of phospho to total (PT) were not associated with karyotpe but were higher in patients with FLT3 mutations. Higher levels of pFOXO3A or PT-FOXO3A were associated with increased proliferation evidenced by strong correlation with higher WBC, percent marrow, and blood blasts and by correlation with higher levels of Cyclins B1, D1 and D3, pGSK3, pMTOR, and pStat5. Patients with High levels of pFOXO3A or PT-FOXO3A had higher rates of primary resistance and shorter remission durations, which combine to cause an inferior survival experience (P = 0.0002). This effect was independent of cytogenetics. PT-FOXO3A was a statistically significant independent predictor in multivariate analysis. Conclusions: High levels of phosphorylation of FOXO3A is a therapeutically targetable, independent adverse prognostic factor in AML. Clin Cancer Res; 16(6); 1865–74

Mechanisms of resistance to decitabine in the myelodysplastic syndrome

Purpose The DNA methylation inhibitor 5-aza-2′-deoxycytidine (DAC) is approved for the treatment of myelodysplastic syndromes (MDS), but resistance to DAC develops during treatment and mechanisms of resistance remain unknown. Therefore, we investigated mechanisms of primary and secondary resistance to DAC in MDS. Patients and Methods We performed Quantitative Real-Time PCR to examine expression of genes related to DAC metabolism prior to therapy in 32 responders and non-responders with MDS as well as 14 patients who achieved a complete remission and subsequently relapsed while on therapy (secondary resistance). We then performed quantitative methylation analyses by bisulfite pyrosequencing of 10 genes as well as Methylated CpG Island Amplification Microarray (MCAM) analysis of global methylation in secondary resistance. Results Most genes showed no differences by response, but the CDA/DCK ratio was 3 fold higher in non-responders than responders (P<.05), suggesting that this could be a mechanism of primary resistance. There were no significant differences at relapse in DAC metabolism genes, and no DCK mutations were detected. Global methylation measured by the LINE1 assay was lower at relapse than at diagnosis (P<.05). On average, the methylation of 10 genes was lower at relapse (16.1%) compared to diagnosis (18.1%) (P<.05).MCAM analysis showed decreased methylation of an average of 4.5% (range 0.6%–9.7%) of the genes at relapse. By contrast, new cytogenetic changes were found in 20% of patients. Conclusion Pharmacological mechanisms are involved in primary resistance to DAC, whereas hypomethylation does not prevent a relapse for patients with DAC treatment.

Snm1B/Apollo mediates replication fork collapse and S Phase checkpoint activation in response to DNA interstrand cross-links

The removal of DNA interstrand cross-links (ICLs) has proven to be notoriously complicated due to the involvement of multiple pathways of DNA repair, which include the Fanconi anemia/BRCA pathway, homologous recombination and components of the nucleotide excision and mismatch repair pathways. Members of the SNM1 gene family have also been shown to have a role in mediating cellular resistance to ICLs, although their precise function has remained elusive. Here, we show that knockdown of Snm1B/Apollo in human cells results in hypersensitivity to mitomycin C (MMC), but not to IR. We also show that Snm1B-deficient cells exhibit a defective S phase checkpoint in response to MMC, but not to IR, and this finding may account for the specific sensitivity to the cross-linking drug. Interestingly, although previous studies have largely implicated ATR as the major kinase activated in response to ICLs, we show that it is activation of the ATM-mediated checkpoint that is defective in Snm1B-deficient cells. The requirement for Snm1B in ATM checkpoint activation specifically after ICL damage is correlated with its role in promoting double-strand break formation, and thus replication fork collapse. Consistent with this result Snm1B was found to interact directly with Mus81-Eme1, an endonuclease previously implicated in fork collapse. In addition, we also show that Snm1B interacts with the Mre11-Rad50-Nbs1 (MRN) complex and with FancD2 further substantiating its role as a checkpoint/DNA repair protein.

Survivin is highly expressed in CD34 +38 - leukemic stem/progenitor cells and predicts poor clinical outcomes in AML

Survivin, a member of the inhibitors of apoptosis protein family, plays important roles in cell proliferation and survival and is highly expressed in various malignancies, including leukemias. To better understand its role in acute myeloid leukemia (AML), we profiled survivin expression in samples obtained from 511 newly diagnosed AML patients and in CD34(+)38(-) AML stem/progenitor cells using a validated reverse-phase protein array; we correlated its levels with clinical outcomes and with levels of other proteins in the same sample set. We found that survivin levels were higher in bone marrow than in paired peripheral blood leukemic cells (n = 140, P = .0001) and that higher survivin levels significantly predicted shorter overall (P = .016) and event-free (P = .023) survival in multivariate Cox model analysis. Importantly, survivin levels were significantly higher in CD34(+)38(-) AML stem/progenitor cells than in bulk blasts and total CD34(+) AML cells (P < .05). Survivin expression correlated with the expressions of multiple proteins involved with cell proliferation and survival. Particularly, its expression strongly correlated with HIF1α in the stem/progenitor cell compartment. These results suggest that survivin is a prognostic biomarker in AML and that survivin, which is overexpressed in AML stem/progenitor cells, remains a potentially important target for leukemia therapy.

Cdc5L interacts with ATR and is required for the S‐phase cell‐cycle checkpoint

Cell division cycle 5‐like protein (Cdc5L) is a core component of the putative E3 ubiquitin ligase complex containing Prp19/Pso4, Plrg1 and Spf27. This complex has been shown to have a role in pre‐messenger RNA splicing from yeast to humans; however, more recent studies have described a function for this complex in the cellular response to DNA damage. Here, we show that Cdc5L interacts physically with the cell‐cycle checkpoint kinase ataxia‐telangiectasia and Rad3‐related (ATR). Depletion of Cdc5L by RNA‐mediated interference methods results in a defective S‐phase cell‐cycle checkpoint and cellular sensitivity in response to replication‐fork blocking agents. Furthermore, we show that Cdc5L is required for the activation of downstream effectors or mediators of ATR checkpoint function such as checkpoint kinase 1 (Chk1), cell cycle checkpoint protein Rad 17 (Rad17) and Fanconi anaemia complementation group D2 protein (FancD2). In addition, we have mapped the ATR‐binding region in Cdc5L and show that a deletion mutant that is unable to interact with ATR is defective in the rescue of the checkpoint deficiency in Cdc5L‐depleted cells. These findings show a new function for Cdc5L in the regulation of the ATR‐mediated cell‐cycle checkpoint in response to genotoxic agents.

Suppression of Peutz-Jeghers Polyposis by Targeting Mammalian Target of Rapamycin Signaling

Purpose: Peutz-Jeghers syndrome (PJS) is a unique disorder characterized by the development of hamartomas in the gastrointestinal tract as well as increased risks for variety of malignancies. Germ-line mutations of LKB1 cause PJS. We have generated Lkb1+/− mice, which model human PJS. Rapamycin and its analogues are promising preventive and therapeutic agents that specifically inhibit signaling from mammalian target of rapamycin (mTOR). Hyperactivation of mTOR signaling has been associated with PJS. The objective of the study is to investigate the efficacy of mTOR inhibition in suppressing Peutz-Jeghers polyposis in Lkb1+/− mice. Experimental Design: We initiated a trial of rapamycin in Lkb1+/− mice at 9 months of age (after the onset of polyposis) at the dose of 2 mg/kg/d for a 2-month period. We assessed the efficacy of rapamycin by measuring polyp sizes and tumor burden. To examine the effect of rapamycin on mTOR signaling, phosphorylation levels of S6 were evaluated by immunostaining. Results: We observed a significant decrease in mean tumor burden (Students t test, P = 0.023) as well as total tumor burden in rapamycin-treated group compared with control group. Comparison of the polyp size observed in both rapamycin-treated and control groups showed that rapamycin efficiently decreased the tumor burden of large polyps (>8 mm). This inhibition of rapamycin was associated with a decrease in phosphorylated S6 levels in the polyps. Conclusions: Rapamycin effectively suppresses Peutz-Jeghers polyposis in a mouse model, suggesting that rapamycin or its analogues may represent a new targeted therapy for the treatment of PJS.

Expression of ARC (apoptosis repressor with caspase recruitment domain), an antiapoptotic protein, is strongly prognostic in AML

Regulators of apoptosis in acute myeloid leukemia (AML) have been extensively studied and are considered excellent therapeutic targets. Apoptosis repressor with caspase recruitment domain (ARC), an antiapoptotic protein originally found to be involved in apoptosis of cardiac cells, was recently demonstrated to be overexpressed in several solid tumors. To assess its importance in AML, we profiled ARC expression in 511 newly diagnosed AML patients using a validated robust reverse-phase protein array and correlated ARC levels with clinical outcomes. ARC was variably expressed in samples from patients with AML. ARC level was not associated with cytogenetic groups or with FLT-3 mutation status. However, patients with low or medium ARC protein levels had significantly better outcomes than those with high ARC levels: longer overall survival (median, 53.9 or 61.6 vs 38.9 weeks, P = .0015) and longer remission duration (median, 97.6 or 44.7 vs 31.1 weeks, P = .0007). Multivariate analysis indicated that ARC was a statistically significant independent predictor of survival in AML (P = .00013). Inhibition of ARC promoted apoptosis and sensitized cytosine arabinoside-induced apoptosis in OCI-AML3 cells. These results suggest that ARC expression levels are highly prognostic in AML and that ARC is a potential therapeutic target in AML.

Abnormal expression of FLI1 protein is an adverse prognostic factor in acute myeloid leukemia

Friend leukemia virus integration 1 (FLI1), an Ets transcription factor family member, is linked to acute myelogenous leukemia (AML) by chromosomal events at the FLI1 locus, but the biologic impact of FLI1 expression on AML is unknown. FLI1 protein expression was measured in 511 newly diagnosed AML patients. Expression was similar in peripheral blood (PB) and BM and higher at diagnosis than at relapse (P = .02). Compared with normal CD34(+) cells, expression in AML was above or below normal in 32% and 5% of patients, respectively. Levels were negatively correlated with an antecedent hematologic disorder (P = .002) but not with age or cytogenetics. Mutated NPM1 (P = .0007) or FLT3-ITD (P < .02) had higher expression. FLI1 levels were negatively correlated with 10 of 195 proteins associated with proliferation and stromal interaction, and positively correlated (R > 0.3) with 19 others. The FLI1 level was not predictive of remission attainment, but patients with low or high FLI1 expression had shorter remission duration (22.6 and 40.3 vs 51.1 weeks, respectively; P = .01) and overall survival (45.2 and 35.4 vs 59.4 weeks, respectively; P = .03). High FLI1 levels were adverse in univariate and multivariate analysis. FLI1 expression is frequently abnormal and prognostically adverse in AML. FLI1 and/or its response genes may be therapeutically targetable to interfere with AML cell biology.