Decitabine enhances anti-CD33 monoclonal antibody BI 836858-mediated natural killer ADCC against AML blasts.

Acute myeloid leukemia (AML) is the most common type of acute leukemia, affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence, novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (gemtuzumab ozogamicin) have been shown to improve overall survival, validating CD33 as a target for antibody-based therapy of AML. Here, we report the in vitro efficacy of BI 836858, a fully human, Fc-engineered, anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858-opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine, 2 hypomethylating agents that show efficacy in older patients, did not compromise BI 836858-induced NK-cell-mediated ADCC. Evaluation of BI 836858-mediated ADCC in serial marrow AML aspirates in patients who received a 10-day course of DAC (pre-DAC, days 4, 11, and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared with pre-DAC treatment. Analysis of ligands to activating receptors (NKG2D) showed significantly increased NKG2D ligand [NKG2DL] expression in day 28 post-DAC samples compared with pre-DAC samples; when NKG2DL receptor was blocked using antibodies, BI 836858-mediated ADCC was significantly decreased, suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML.

SPARC promotes leukemic cell growth and predicts acute myeloid leukemia outcome.

Aberrant expression of the secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) gene, which encodes a matricellular protein that participates in normal tissue remodeling, is associated with a variety of diseases including cancer, but the contribution of SPARC to malignant growth remains controversial. We previously reported that SPARC was among the most upregulated genes in cytogenetically normal acute myeloid leukemia (CN-AML) patients with gene-expression profiles predictive of unfavorable outcome, such as mutations in isocitrate dehydrogenase 2 (IDH2-R172) and overexpression of the oncogenes brain and acute leukemia, cytoplasmic (BAALC) and v-ets erythroblastosis virus E26 oncogene homolog (ERG). In contrast, SPARC was downregulated in CN-AML patients harboring mutations in nucleophosmin (NPM1) that are associated with favorable prognosis. Based on these observations, we hypothesized that SPARC expression is clinically relevant in AML. Here, we found that SPARC overexpression is associated with adverse outcome in CN-AML patients and promotes aggressive leukemia growth in murine models of AML. In leukemia cells, SPARC expression was mediated by the SP1/NF-κB transactivation complex. Furthermore, secreted SPARC activated the integrin-linked kinase/AKT (ILK/AKT) pathway, likely via integrin interaction, and subsequent ß-catenin signaling, which is involved in leukemia cell self-renewal. Pharmacologic inhibition of the SP1/NF-κB complex resulted in SPARC downregulation and leukemia growth inhibition. Together, our data indicate that evaluation of SPARC expression has prognosticative value and SPARC is a potential therapeutic target for AML.

Epigenetics meets genetics in acute myeloid leukemia: clinical impact of a novel seven-gene score.

PURPOSE: Molecular risk stratification of acute myeloid leukemia (AML) is largely based on genetic markers. However, epigenetic changes, including DNA methylation, deregulate gene expression and may also have prognostic impact. We evaluated the clinical relevance of integrating DNA methylation and genetic information in AML. METHODS: Next-generation sequencing analysis of methylated DNA identified differentially methylated regions (DMRs) associated with prognostic mutations in older (≥ 60 years) cytogenetically normal (CN) patients with AML (n = 134). Genes with promoter DMRs and expression levels significantly associated with outcome were used to compute a prognostic gene expression weighted summary score that was tested and validated in four independent patient sets (n = 355). RESULTS: In the training set, we identified seven genes (CD34, RHOC, SCRN1, F2RL1, FAM92A1, MIR155HG, and VWA8) with promoter DMRs and expression associated with overall survival (OS; P ≤ .001). Each gene had high DMR methylation and lower expression, which were associated with better outcome. A weighted summary expression score of the seven gene expression levels was computed. A low score was associated with a higher complete remission (CR) rate and longer disease-free survival and OS (P < .001 for all end points). This was validated in multivariable models and in two younger (< 60 years) and two older independent sets of patients with CN-AML. Considering the seven genes individually, the fewer the genes with high expression, the better the outcome. Younger and older patients with no genes or one gene with high expression had the best outcomes (CR rate, 94% and 87%, respectively; 3-year OS, 80% and 42%, respectively). CONCLUSION: A seven-gene score encompassing epigenetic and genetic prognostic information identifies novel AML subsets that are meaningful for treatment guidance.

GAS6 expression identifies high-risk adult AML patients: potential implications for therapy.

Emerging data demonstrate important roles for the TYRO3/AXL/MERTK receptor tyrosine kinase (TAM RTK) family in diverse cancers. We investigated the prognostic relevance of GAS6 expression, encoding the common TAM RTK ligand, in 270 adults (n=71 aged<60 years; n=199 aged ⩾60 years) with de novo cytogenetically normal acute myeloid leukemia (CN-AML). Patients expressing GAS6 (GAS6+), especially those aged ⩾60 years, more often failed to achieve a complete remission (CR). In all patients, GAS6+ patients had shorter disease-free (DFS) and overall (OS) survival than patients without GAS6 expression (GAS6-). After adjusting for other prognostic markers, GAS6+ predicted CR failure (P=0.02), shorter DFS (P=0.004) and OS (P=0.04). To gain further biological insights, we derived a GAS6-associated gene-expression signature (P<0.001) that in GAS6+ patients included overexpressed BAALC and MN1, known to confer adverse prognosis in CN-AML, and overexpressed CXCL12, encoding stromal cell-derived factor, and its receptor genes, chemokine (C-X-C motif) receptor 4 (CXCR4) and CXCR7. This study reports for the first time that GAS6 expression is an adverse prognostic marker in CN-AML. Although GAS6 decoy receptors are not yet available in the clinic for GAS6+ CN-AML therapy, potential alternative therapies targeting GAS6+-associated pathways, for example, CXCR4 antagonists, may be considered for GAS6+ patients to sensitize them to chemotherapy.

Eradicating acute myeloid leukemia in a Mll(PTD/wt):Flt3(ITD/wt) murine model: a path to novel therapeutic approaches for human disease.

The coexpression of the MLL partial tandem duplication (PTD) and the FLT3 internal tandem duplication (ITD) mutations associate with a poor outcome in cytogenetically normal acute myeloid leukemia (AML). In mice, a double knock-in (dKI) of Mll(PTD/wt) and Flt3(ITD/wt) mutations induces spontaneous AML with an increase in DNA methyltransferases (Dnmt1, 3a, and 3b) and global DNA methylation index, thereby recapitulating its human AML counterpart. We determined that a regulator of Dnmts, miR-29b, is downregulated in bone marrow of dKI AML mice. Bortezomib exerted a dose-dependent increase in miR-29b expression in AML blasts ex vivo, followed by decreased Dnmts, reduced proliferation, and increased apoptosis. In vivo, bortezomib was not active against dKI AML, yet liposomal-encapsulated bortezomib, as a single agent, reversed downregulation of miR-29b in vivo and induced a long-term (90-day) disease-free remission in 80% of dKI AML mice that exhibited high leukemic burden at the start of therapy, yet showed no signs of relapse at autopsy. Taken together, these data support that liposomal bortezomib, as a single agent, eradicates Mll(PTD/wt):Flt3(ITD/wt) AML in mouse and may represent a powerful and potentially curative approach to high-risk human disease.

Clinical role of microRNAs in cytogenetically normal acute myeloid leukemia: miR-155 upregulation independently identifies high-risk patients.

PURPOSE: To evaluate the impact of miR-155 on the outcome of adults with cytogenetically normal (CN) acute myeloid leukemia (AML) in the context of other clinical and molecular prognosticators and to gain insight into the leukemogenic role of this microRNA. PATIENTS AND METHODS: We evaluated 363 patients with primary CN-AML. miR-155 levels were measured in pretreatment marrow and blood by NanoString nCounter assays that quantified the expression of the encoding gene MIR155HG. All molecular prognosticators were assessed centrally. miR-155-associated gene and microRNA expression profiles were derived using microarrays. RESULTS: Considering all patients, high miR-155 expression was associated with a lower complete remission (CR) rate (P < .001) and shorter disease-free survival (P = .001) and overall survival (OS; P < .001) after adjusting for age. In multivariable analyses, high miR-155 expression remained an independent predictor for a lower CR rate (P = .007) and shorter OS (P < .001). High miR-155 expressers had approximately 50% reduction in the odds of achieving CR and 60% increase in the risk of death compared with low miR-155 expressers. Although high miR-155 expression was not associated with a distinct microRNA expression profile, it was associated with a gene expression profile enriched for genes involved in cellular mechanisms deregulated in AML (eg, apoptosis, nuclear factor-κB activation, and inflammation), thereby supporting a pivotal and unique role of this microRNA in myeloid leukemogenesis. CONCLUSION: miR-155 expression levels are associated with clinical outcome independently of other strong clinical and molecular predictors. The availability of emerging compounds with antagonistic activity to microRNAs in the clinic provides the opportunity for future therapeutic targeting of miR-155 in AML.

Inhibition of the receptor tyrosine kinase Axl impedes activation of the FLT3 internal tandem duplication in human acute myeloid leukemia: implications for Axl as a potential therapeutic target.

Approximately 20% to 25% of patients with acute myeloid leukemia (AML) have a constitutively activated FLT3-internal tandem duplication (FLT3-ITD), and these patients exhibit a poor prognosis. Here, we report that Axl, a receptor tyrosine kinase (RTK) overexpressed and constitutively active in human AML, targets the RTK FLT3 in FLT3-ITD(+) AML. Abrogation of Axl activation by soluble Axl chimeric protein (Axl-Fc) or small interfering RNA (siRNA) diminishes constitutive FLT3 phosphorylation in FLT3-ITD(+) AML. In addition, inhibition of Axl activation by Axl-Fc interferes with the physical interaction between Axl and FLT3. We found that Axl-Fc, a pharmacologic Axl inhibitor, or siRNA targeting Axl inhibits cell growth, induces cell-cycle arrest and apoptosis, and relieves a block in myeloid differentiation of FLT3-ITD(+) AML in vitro. Axl-Fc also suppresses the growth of human FLT3-ITD(+) AML in vivo. Collectively, our data suggest that Axl contributes to the pathogenesis of FLT3-ITD(+) AML through, at least in part, positive regulation of constitutive FLT3 activation. This also suggests that Axl should be pursued as a potential target for the treatment of FLT3-ITD(+) AML.

Lenalidomide-mediated enhanced translation of C/EBPα-p30 protein up-regulates expression of the antileukemic microRNA-181a in acute myeloid leukemia.

Recently, we showed that increased miR-181a expression was associated with improved outcomes in cytogenetically normal acute myeloid leukemia (CN-AML). Interestingly, miR-181a expression was increased in CN-AML patients harboring CEBPA mutations, which are usually biallelic and associate with better prognosis. CEBPA encodes the C/EBPα transcription factor. We demonstrate here that the presence of N-terminal CEBPA mutations and miR-181a expression are linked. Indeed, the truncated C/EBPα-p30 isoform, which is produced from the N-terminal mutant CEBPA gene or from the differential translation of wild-type CEBPA mRNA and is commonly believed to have no transactivation activity, binds to the miR-181a-1 promoter and up-regulates the microRNA expression. Furthermore, we show that lenalidomide, a drug approved for myelodysplastic syndromes and multiple myeloma, enhances translation of the C/EBPα-p30 isoform, resulting in higher miR-181a levels. In xenograft mouse models, ectopic miR-181a expression inhibits tumor growth. Similarly, lenalidomide exhibits antitumorigenic activity paralleled by increased miR-181a expression. This regulatory pathway may explain an increased sensitivity to apoptosis-inducing chemotherapy in subsets of AML patients. Altogether, our data provide a potential explanation for the improved clinical outcomes observed in CEBPA-mutated CN-AML patients, and suggest that lenalidomide treatment enhancing the C/EBPα-p30 protein levels and in turn miR-181a may sensitize AML blasts to chemotherapy.