Anna Staffas

Presence of FLT3-ITD and high BAALC expression are independent prognostic markers in childhood acute myeloid leukemia

Mutation status of FLT3, NPM1, CEBPA, and WT1 genes and gene expression levels of ERG, MN1, BAALC, FLT3, and WT1 have been identified as possible prognostic markers in acute myeloid leukemia (AML). We have performed a thorough prognostic evaluation of these genetic markers in patients with pediatric AML enrolled in the Nordic Society of Pediatric Hematology and Oncology (NOPHO) 1993 or NOPHO 2004 protocols. Mutation status and expression levels were analyzed in 185 and 149 patients, respectively. Presence of FLT3-internal tandem duplication (ITD) was associated with significantly inferior event-free survival (EFS), whereas presence of an NPM1 mutation in the absence of FLT3-ITD correlated with significantly improved EFS. Furthermore, high levels of ERG and BAALC transcripts were associated with inferior EFS. No significant correlation with survival was seen for mutations in CEBPA and WT1 or with gene expression levels of MN1, FLT3, and WT1. In multivariate analysis, the presence of FLT3-ITD and high BAALC expression were identified as independent prognostic markers of inferior EFS. We conclude that analysis of the mutational status of FLT3 and NPM1 at diagnosis is important for prognostic stratification of patients with pediatric AML and that determination of the BAALC gene expression level can add valuable information.

Prognostic implications of mutations in NOTCH1 and FBXW7 in childhood T-all treated according to the NOPHO ALL-1992 and ALL-2000 protocols

In children, T‐cell acute lymphoblastic leukemia (T‐ALL) has inferior prognosis compared with B‐cell precursor ALL. In order to improve survival, individualized treatment strategies and thus risk stratification algorithms are warranted, ideally already at the time of diagnosis.

Loss of One Copy of Zfp148 Reduces Lesional Macrophage Proliferation and Atherosclerosis in Mice by Activating p53

Rationale: Cell proliferation and cell cycle control mechanisms are thought to play central roles in the pathogenesis of atherosclerosis. The transcription factor Zinc finger protein 148 (Zfp148) was shown recently to maintain cell proliferation under oxidative conditions by suppressing p53, a checkpoint protein that arrests proliferation in response to various stressors. It is established that inactivation of p53 accelerates atherosclerosis, but whether increased p53 activation confers protection against the disease remains to be determined. Objective: We aimed to test the hypothesis that Zfp148 deficiency reduces atherosclerosis by unleashing p53 activity. Methods and Results: Mice harboring a gene-trap mutation in the Zfp148 locus (Zfp148gt/+) were bred onto the apolipoprotein E (Apoe)–/– genetic background and fed a high-fat or chow diet. Loss of 1 copy of Zfp148 markedly reduced atherosclerosis without affecting lipid metabolism. Bone marrow transplantation experiments revealed that the effector cell is of hematopoietic origin. Peritoneal macrophages and atherosclerotic lesions from Zfp148gt/+Apoe–/– mice showed increased levels of phosphorylated p53 compared with controls, and atherosclerotic lesions contained fewer proliferating macrophages. Zfp148gt/+Apoe–/– mice were further crossed with p53-null mice (Trp53–/– [the gene encoding p53]). There was no difference in atherosclerosis between Zfp148gt/+Apoe–/– mice and controls on a Trp53+/– genetic background, and there was no difference in levels of phosphorylated p53 or cell proliferation. Conclusions: Zfp148 deficiency increases p53 activity and protects against atherosclerosis by causing proliferation arrest of lesional macrophages, suggesting that drugs targeting macrophage proliferation may be useful in the treatment of atherosclerosis.

MiR-139-5p is a potent tumor suppressor in adult acute myeloid leukemia.

Hematopoiesis depends on a tightly controlled balance of selfrenewal, proliferation, cell death and differentiation. Although the disturbance of this equilibrium creates a predisposition to leukemogenesis, targeted manipulation or modulation can in turn lead to therapeutic advances. In addition to chromosomal aberrations and frequently mutated genes such as NPM1 and FLT3, dysregulation of microRNAs (miRNAs) is now recognized as having an important role in leukemogenesis. Distinct miRNA expression profiles can classify AML subgroups and miRNAs have recently emerged as novel therapeutic targets in hematopoietic malignancies. Building on our previous efforts to resolve potential involvement of miRNAs in hematopoiesis, we identified miR-139-5p as a myeloid-specific miRNA with expression being restricted to neutrophils and macrophages (Supplementary Figure S1A). This distinct expression profile during normal hematopoiesis pointed towards a possible deregulation in malignant hematopoiesis. In order to relate miR-139-5p expression to distinct genetic subgroups and clinical outcome in adult AML patients, we analyzed the published miRNA sequencing dataset from The Cancer Genome Atlas (TCGA). A trend for prolonged overall survival (OS) was observed for patients with miR-139-5p levels above the median independent of cytogenetic subgroup (P= 0.07, Figure 1a left panel), whereas AML patients with normal karyotype (CN-AML) exhibited a significantly better OS (P= 0.02, Figure 1a right panel). Our findings were further corroborated in CN-AML by comparing the expression quartiles, demonstrating that low miR-139-5p expressors (q3 and q4) are associated with an unfavorable OS (Supplementary Figure S1B). In addition, no effect could be demonstrated for the less abundant miR-139-3p (Supplementary Figure S1C), indicating that miR-139-5p is the active strand. Our findings are in line with Emmrich et al., who associated high miR-139-5p levels with a favorable outcome in pediatric AML. Further analysis of the TCGA dataset revealed a significant downregulation of miR-139-5p in CN-AML patients harboring mutated FLT3 (P= 0.03 and P= 0.001, respectively) compared with the total AML population. (Figure 1b left panel). Considering the poor prognosis of CN-AML patients with mutated FLT3, our findings reinforce the unfavorable outcome of CN-AML patients with low miR-139-5p levels. In contrast, miR-139-5p was upregulated in patients carrying an inv(16) (P= 0.001) translocation, a prognostically favorable AML subgroup (Figure 1b left panel). No differences were found for miR-139-5p levels in AML patients with t(15;17) and MLL rearrangements, indicating that miR-139-5p levels are associated with specific genetic subtypes rather than with the degree of differentiation. These findings were validated by qRT-PCR in an independent cohort consisting of 49 adult AML patients and 4 healthy donors of total bone marrow (bm) referred to as the Ulm cohort with uniform treatment procedure. All patient samples from the Ulm cohort were collected from adult patients enroled on German-Austrian AML Study Group (AMLSG) treatment protocols for younger adults (AMLSG-HD98A (NCT00146120) and AMLSG 07-04 (NCT00151242)) and comprised Ficoll gradient purified mononuclear cells mainly from diagnostic bm samples with blast counts of ~ 80% in all analyzed cases. In line with the TCGA dataset, patients harboring mutated FLT3 displayed the lowest miR-139-5p expression among the various cytogenetic subtypes in the Ulm cohort (P= 0.03, Figure 1b right panel). Decreased expression of miR-139 in NPM1-mutated CNAML patients, has been previously reported by Garzon et al. We observed a similar trend within the TCGA dataset (P= 0.06) (Figure 1b left panel), highlighting the robustness of both the datasets. Taken together, we have demonstrated in two independent AML patient cohorts that decreased miR-139-5p levels are associated with mutated FLT3 and with a lower OS in CN-AML in the TCGA dataset. However, it remains to be determined if miR-139-5p is directly or indirectly transcriptionally regulated through activated FLT3. The distinct expression patterns of miR-139-5p in CN-AML imply a tumor suppressor role for this miRNA. In order to further explore the therapeutic potential of miR-139-5p, we used a syngenic bm transplantation AML model based on the transformation of murine bm cells through the combined retroviral overexpression of Hoxa9 and Meis1 (Hoxa9/Meis1), which causes a rapid AML in vivo compared with the pre-leukemic Hoxa9 cells that lead to a long latency AML. Upregulation of HOXA9 and MEIS1 is frequently observed in NPM1-mutated CN-AML and enhances FLT3 signaling. Based on the expression pattern of miR-139-5p in AML patients, we further hypothesized that miR-139-5p would be downregulated in Hoxa9/Meis1 cells compared with untransformed wild-type bm cells. Indeed, miR-139-5p was significantly downregulated in Hoxa9/Meis1 cells compared with untransduced bm cells as measured by qRT-PCR (Supplementary Figure S1D), highlighting the relevance of this in vivo model. To validate the putative tumor suppressor function of miR-139-5p, its expression levels were restored through ectopic lentiviral coexpression of miR-139 and GFP as reported in Hoxa9/Meis1-transformed bm cells. Hoxa9/Meis1/miR-139-5p bm or Hoxa9/Meis1 cells overexpressing an empty control vector (miR-ctrl) were FACS-sorted for GFP, followed by transplantation of 2 × 10 cells per arm into lethally irradiated recipient mice. Restoration of miR-139 levels significantly delayed Hoxa9/Meis1-mediated leukemogenesis (P= 0.0003, Figure 2a) in two independent experiments, highlighting its tumor suppressor activity in a primary transplantation model. Bm cells of deceased mice retained overexpression of miR-139-5p compared with the control arm as quantified by qRT-PCR (Supplementary Figure S1E). Lentiviral expression of miR-139-5p in Hoxa9/Meis1 cells restored miR-139-5p at similar levels compared with untransduced bm cells (Supplementary Figures 1D and E), minimizing possible artefacts of ectopic expression. Notably, we detected no endogenous expression of miR-139-3p in the Hoxa9/Meis1/miR-ctrl bm and overexpression of miR-139 generated lower levels of miR-139-3p in comparison with miR-139-5p, highlighting miR-139-5p as the active strand (Supplementary Figure S1F). Recently, Gibbs et al. demonstrated that Hoxa9/Meis1 cells harbor three, immunophenotypically distinct compartments with varying tumor-initiating activity. Therefore, bm cells of deceased mice were analyzed by flow cytometry for their respective immunophenotype (Figure 2b). We found a significant reduction Citation: Blood Cancer Journal (2016) 6, e508; doi:10.1038/bcj.2016.110

MicroRNA-155 is upregulated in MLL-rearranged AML but its absence does not affect leukemia development

MicroRNA-155 (miR-155) is an oncogenic miRNA upregulated in various tumor types and leukemias and has been suggested as a potential drug target. Based on our previous work detecting high miR-155 levels in response to Meis1 overexpression in a murine Hox leukemia model, we show here the relationship among HOXA9, MEIS1, and miR-155 levels in MLL-translocated acute myeloid leukemia (AML) patients. Using mouse bone marrow cells transformed by MLL-fusion genes expressing graduated levels of Meis1, we show a positive correlation between miR-155 and Meis1. However, using a miR-155-knockout mouse model, we show that the absence and the depletion of miR-155 have no effect on leukemia formation or progression. We also show for the first time that miR-155 levels are correlated with MLL translocations, but that miR-155 expression is dispensable for the formation of AML and has no effect on leukemia progression.

MicroRNA-155 is a direct target of Meis1, but not a driver in acute myeloid leukemia

Micro-ribonucleic acid-155 (miR-155) is one of the first described oncogenic miRNAs. Although multiple direct targets of miR-155 have been identified, it is not clear how it contributes to the pathogenesis of acute myeloid leukemia. We found miR-155 to be a direct target of Meis1 in murine Hoxa9/Meis1 induced acute myeloid leukemia. The additional overexpression of miR-155 accelerated the formation of acute myeloid leukemia in Hoxa9 as well as in Hoxa9/Meis1 cells in vivo. However, in the absence or following the removal of miR-155, leukemia onset and progression were unaffected. Although miR-155 accelerated growth and homing in addition to impairing differentiation, our data underscore the pathophysiological relevance of miR-155 as an accelerator rather than a driver of leukemogenesis. This further highlights the complexity of the oncogenic program of Meis1 to compensate for the loss of a potent oncogene such as miR-155. These findings are highly relevant to current and developing approaches for targeting miR-155 in acute myeloid leukemia.

Zinc finger protein 148 is dispensable for primitive and definitive hematopoiesis in mice.

Hematopoiesis is regulated by transcription factors that induce cell fate and differentiation in hematopoietic stem cells into fully differentiated hematopoietic cell types. The transcription factor zinc finger protein 148 (Zfp148) interacts with the hematopoietic transcription factor Gata1 and has been implicated to play an important role in primitive and definitive hematopoiesis in zebra fish and mouse chimeras. We have recently created a gene-trap knockout mouse model deficient for Zfp148, opening up for analyses of hematopoiesis in a conventional loss-of-function model in vivo. Here, we show that Zfp148-deficient neonatal and adult mice have normal or slightly increased levels of hemoglobin, hematocrit, platelets and white blood cells, compared to wild type controls. Hematopoietic lineages in bone marrow, thymus and spleen from Zfp148 gt/gt mice were further investigated by flow cytometry. There were no differences in T-cells (CD4 and CD8 single positive cells, CD4 and CD8 double negative/positive cells) in either organ. However, the fraction of CD69- and B220-positive cells among lymphocytes in spleen was slightly lower at postnatal day 14 in Zfp148 gt/gt mice compared to wild type mice. Our results demonstrate that Zfp148-deficient mice generate normal mature hematopoietic populations thus challenging earlier studies indicating that Zfp148 plays a critical role during hematopoietic development.

The Endothelin receptor type A is a downstream target of Hoxa9 and Meis1 in Acute Myeloid Leukemia

Endothelin receptor type A (EDNRA) is known as a mediator of cell proliferation and survival. Aberrant regulation of EDNRA has been shown to play a role in tumor growth and metastasis. Using a global gene expression screen, we found that expression of Ednra was upregulated in murine leukemia inducing cells co-expressing Hoxa9 and Meis1 compared to cells only expressing Hoxa9. The aim of this study was to explore the role of Ednra in leukemogenesis further. In a murine bone marrow transplantation model, mice transplanted with cells overexpressing Ednra and Hoxa9 succumbed to leukemia significantly earlier than mice transplanted with cells overexpressing Hoxa9 only. Furthermore, overexpression of Ednra led to increased proliferation and resistance to apoptosis of bone marrow cells in vitro. We could also show that Meis1 binds to the Ednra promoter region, suggesting a regulatory role for Meis1 in Ednra expression. Taken together, our results suggest a role for Ednra in Hoxa9/Meis1-driven leukemogenesis.

Upregulation of Flt3 is a passive event in Hoxa9/Meis1-induced acute myeloid leukemia in mice.

HOXA9, MEIS1 and FLT3 are genes frequently upregulated in human acute myeloid leukemia. Hoxa9 and Meis1 also cooperate to induce aggressive AML with high Flt3 expression in mice, suggesting an important role for Flt3 in Hoxa9/Meis1-induced leukemogenesis. To define the role of Flt3 in AML with high Hoxa9/Meis1, we treated mice with Hoxa9/Meis1-induced AML with the Flt3 inhibitor AC220, used an Flt3-ligand (FL−/−) knockout model, and investigated whether overexpression of Flt3 could induce leukemia together with overexpression of Hoxa9. Flt3 inhibition by AC220 did not delay AML development in mice transplanted with bone marrow cells overexpressing Hoxa9 and Meis1. In addition, Hoxa9/Meis1 cells induced AML in FL−/− mice as rapid as in wild-type mice. However, FL−/− mice had reduced organ infiltration compared with wild-type mice, suggesting some Flt3-dependent effect on leukemic invasiveness. Interestingly, leukemic Hoxa9/Meis1 cells from sick mice expressed high levels of Flt3 regardless of presence of its ligand, showing that Flt3 is a passive marker on these cells. In line with this, combined engineered overexpression of Flt3 and Hoxa9 did not accelerate the progression to AML. We conclude that the Hoxa9- and Meis1-associated upregulation of Flt3 is not a requirement for leukemic progression induced by Hoxa9 and Meis1.