Yihua Qiu

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.

BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies

Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response.

Different Changes in Protein and Phosphoprotein Levels Result from Serum Starvation of High-Grade Glioma and Adenocarcinoma Cell Lines

Tumor cells undergoing serum starvation in vitro partially mimic metabolically stressed cells trying to adjust to a changed environment in vivo by inducing signal transduction and gene expression so that the tumor continues to grow. Our hypothesis is that the changes in protein and phosphoprotein levels after serum starvation may reflect the adapted phenotype of the tumor, which could be targeted for therapy. We used reverse-phase protein microarrays to interrogate five high-grade glioma cell lines and seven adenocarcinoma cell lines for differences in the level of 81 proteins and 25 phosphoproteins. All cell lines were studied in the well-fed condition of growth with 10% FBS and the starved condition of 0.5% FBS. Protein expression levels were normalized to beta-actin and trichotomized as increased (+1, upper 75th quartile), decreased (-1, lowest 25th quartile), or unchanged (0, others) to focus on the patterns of the biggest (and hopefully most robust) changes in protein and phosphoprotein levels. We examined these trichotomized values to better understand Starved-Fed differences among the cell lines and thereby gain better/clearer insight into the effects of serum starvation on potential cellular responses. In general, the expression of proteins and phosphoproteins 24 h after FBS starvation increased more often in glioma lines than in adenocarcinoma lines, which appeared to have fewer increased protein scores and more decreased scores. Many of the proteins increased in gliomas were downstream targets of the PTEN-PI-3 kinase-AKT, EGFR-MAPK-Stat, and transcription activator-polyamine signaling pathways. In adenocarcinomas, the expression of proteins and phosphoproteins generally increased in apoptosis pathways, while there were minor fluctuations in the other pathways above. Contrawise, gliomas become resistant to apoptosis after 24 h of serum starvation and upregulate transcription activators and polyamines more so than adenocarciomas.

PI3K/AKT pathway activation in acute myeloid leukaemias is not associated with AKT1 pleckstrin homology domain mutation

Despite its’ central role, the precise mechanisms of the phosphoinositide 3‐kinase/Akt (PI3K)/Akt pathway activation in acute myeloid leukaemia (AML) have not been elucidated. Recently, a recurrent novel AKT1 pleckstrin homology domain (PHD) mutation leading to membrane translocation, constitutive AKT activation and leukaemia development in mice was described. To assess AKT1 PHD mutations in AML, we sequenced 57 specimens from 49 AML patients, all of whom showed PI3K/AKT pathway activation by analysis of total and phospho‐protein expression for AKT, mTor, p70S6Kinase, S6ribosomal protein and PTEN. No mutations in AKT1 PHD were identified, making this mutation an unlikely cause of PI3K/AKT pathway activation in AML.

Synergistic Targeting of AML Stem/Progenitor Cells With IAP Antagonist Birinapant and Demethylating Agents

BACKGROUND Acute myeloid leukemia (AML) therapy has limited long-term efficacy because patients frequently develop disease relapse because of the inability of standard chemotherapeutic agents to target AML stem/progenitor cells. Here, we identify deregulated apoptotic components in AML stem/progenitor cells and investigate the individual and combinatorial effects of the novel inhibitor of apoptosis (IAP) protein antagonist and second mitochondrial-derived activator of caspases (SMAC) mimetic birinapant and demethylating epigenetic modulators. METHODS Protein expression was measured by reversed-phase protein array in AML patient (n = 511) and normal (n = 21) samples and by western blot in drug-treated cells. The antileukemic activity of birinapant and demethylating agents was assessed in vitro and in an in vivo AML mouse xenograft model (n = 10 mice per group). All statistical tests were two-sided. RESULTS Compared with bulk AML cells, CD34(+)38(-) AML stem/progenitors expressed increased cIAP1 and caspase-8 levels and decreased SMAC levels (one-way analysis of variance followed by Tukeys multiple comparison test, P < .001). Birinapant induced death receptor-/caspase-8-mediated apoptosis in AML cells, including in AML stem/progenitor cells, but not in normal CD34(+) cells. Demethylating agents modulated extrinsic apoptosis pathway components and, when combined with birinapant, were highly synergistic in vitro (combination index < 1), and also more effective in vivo (P < .001, by Student t test, for the median survival of birinapant plus 5-azacytadine vs birinapant alone or vs controls). CONCLUSIONS cIAP1, SMAC, and caspase-8 appear to play a role in AML stem cell survival, and synergistic targeting of these cells with birinapant and demethylating agents shows potential utility in leukemia therapy.

Role of Peroxisome Proliferator-Activated Receptor-γ and Its Coactivator DRIP205 in Cellular Responses to CDDO (RTA-401) in Acute Myelogenous Leukemia

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear receptor (NR) family of transcription factors with important regulatory roles in cellular growth, differentiation, and apoptosis. Using proteomic analysis, we showed expression of PPARgamma protein in a series of 260 newly diagnosed primary acute myelogenous leukemia (AML) samples. Forced expression of PPARgamma enhanced the sensitivity of myeloid leukemic cells to apoptosis induced by PPARgamma agonists 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and 15-deoxy-(12,14)-15DPGJ(2), through preferential cleavage of caspase-8. No effects on cell cycle distribution or differentiation were noted, despite prominent induction of p21 in PPARgamma-transfected cells. In turn, antagonizing PPARgamma function by small interfering RNA or pharmacologic PPARgamma inhibitor significantly diminished apoptosis induction by CDDO. Overexpression of coactivator protein DRIP205 resulted in enhanced differentiation induction by CDDO in AML cells through PPARgamma activation. Studies with DRIP205 deletion constructs showed that the NR boxes of DRIP205 are not required for this coactivation. In a phase I clinical trial of CDDO (RTA-401) in leukemia, CDDO induced an increase in PPARgamma mRNA expression in six of nine patient samples; of those, induction of differentiation was documented in four patients and that of p21 in three patients, all expressing DRIP205 protein. In summary, these findings suggest that cellular levels of PPARgamma regulate induction of apoptosis via caspase-8 activation, whereas the coactivator DRIP205 is a determinant of induction of differentiation, in response to PPARgamma agonists in leukemic cells.

MLL-Rearranged Acute Lymphoblastic Leukemias Activate BCL-2 through H3K79 Methylation and Are Sensitive to the BCL-2-Specific Antagonist ABT-199

Summary Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL) mutations such as the t(4;11) translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11) translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11) patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11) cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias.

p53 pathway dysfunction is highly prevalent in acute myeloid leukemia independent of TP53 mutational status

TP53 mutations are associated with the lowest survival rates in acute myeloid leukemia (AML). In addition to mutations, loss of p53 function can arise via aberrant expression of proteins that regulate p53 stability and function. We examined a large AML cohort using proteomics, mutational profiling and network analyses, and showed that (1) p53 stabilization is universal in mutant TP53 samples, it is frequent in samples with wild-type TP53, and in both cases portends an equally dismal prognosis; (2) the p53 negative regulator Mdm2 is frequently overexpressed in samples retaining wild-type TP53 alleles, coupled with absence of p21 expression and dismal prognosis similar to that of cases with p53 stabilization; (3) AML samples display unique patterns of p53 pathway protein expression, which segregate prognostic groups with distinct cure rates; (4) such patterns of protein activation unveil potential AML vulnerabilities that can be therapeutically exploited.

The protein phosphatase 2A regulatory subunit B55α is a modulator of signaling and microRNA expression in acute myeloid leukemia cells

We recently discovered that the protein phosphatase 2A (PP2A) B55α subunit (PPP2R2A) is under-expressed in primary blast cells and is unfavorable for remission duration in AML patients. In this study, reverse phase protein analysis (RPPA) of 230 proteins in 511 AML patient samples revealed a strong correlation of B55α with a number of proteins including MYC, PKC α, and SRC. B55α suppression in OCI-AML3 cells by shRNA demonstrated that the B subunit is a PKCα phosphatase. B55α does not target SRC, but rather the kinase suppresses protein expression of the B subunit. Finally, the correlation between B55α and MYC levels reflected a complex stoichiometric competition between B subunits. Loss of B55α in OCI-AML3 cells did not change global PP2A activity and the only isoform that is induced is the one containing B56α. In cells containing B55α shRNA, MYC was suppressed with concomitant induction of the competing B subunit B56α (PPP2R5A). A recent study determined that FTY-720, a drug whose action involves the activation of PP2A, resulted in the induction of B55α In AML cells, and a reduction of the B subunit rendered these cells resistant to FTY-720. Finally, reduction of the B subunit resulted in an increase in the expression of miR-191-5p and a suppression of miR-142-3p. B55α regulation of these miRs was intriguing as high levels of miR-191 portend poor survival in AML, and miR-142-3p is mutated in 2% of AML patient samples. In summary, the suppression of B55α activates signaling pathways that could support leukemia cell survival.

Focal Adhesion Kinase as a Potential Target in AML and MDS.

Although overexpression/activation of focal adhesion kinase (FAK) is widely known in solid tumors to control cell growth, survival, invasion, metastasis, gene expression, and stem cell self-renewal, its expression and function in myeloid leukemia are not well investigated. Using reverse-phase protein arrays in large cohorts of newly diagnosed acute myeloid leukemia (AML) and myeloid dysplastic syndrome (MDS) samples, we found that high FAK expression was associated with unfavorable cytogenetics (P = 2 × 10−4) and relapse (P = 0.02) in AML. FAK expression was significantly lower in patients with FLT3-ITD (P = 0.0024) or RAS (P = 0.05) mutations and strongly correlated with p-SRC and integrinβ3 levels. FAK protein levels were significantly higher in CD34+ (P = 5.42 × 10−20) and CD34+CD38− MDS (P = 7.62 × 10−9) cells compared with normal CD34+ cells. MDS patients with higher FAK in CD34+ cells tended to have better overall survival (P = 0.05). FAK expression was significantly higher in MDS patients who later transformed to compared with those who did not transform to AML and in AML patients who transformed from MDS compared with those with de novo AML. Coculture with mesenchymal stromal cells (MSC) increased FAK expression in AML cells. Inhibition of FAK decreased MSC-mediated adhesion/migration and viability of AML cells and prolonged survival in an AML xenograft murine model. Our results suggest that FAK regulates leukemia–stromal interactions and supports leukemia cell survival; hence, FAK is a potential therapeutic target in myeloid leukemia. Mol Cancer Ther; 16(6); 1133–44. ©2017 AACR.