A noncanonical enzymatic function of PIWIL4 maintains genomic integrity and leukemic growth in AML.

RNA-binding proteins (RBPs) form a large and diverse class of factors, many members of which are overexpressed in hematologic malignancies. RBPs participate in various processes of messenger RNA (mRNA) metabolism and prevent harmful DNA:RNA hybrids or R-loops. Here, we report that PIWIL4, a germ stem cell-associated RBP belonging to the RNase H-like superfamily, is overexpressed in patients with acute myeloid leukemia (AML) and is essential for leukemic stem cell function and AML growth, but dispensable for healthy human hematopoietic stem cells. In AML cells, PIWIL4 binds to a small number of known piwi-interacting RNA. Instead, it largely interacts with mRNA annotated to protein-coding genic regions and enhancers that are enriched for genes associated with cancer and human myeloid progenitor gene signatures. PIWIL4 depletion in AML cells downregulates the human myeloid progenitor signature and leukemia stem cell (LSC)-associated genes and upregulates DNA damage signaling. We demonstrate that PIWIL4 is an R-loop resolving enzyme that prevents R-loop accumulation on a subset of AML and LSC-associated genes and maintains their expression. It also prevents DNA damage, replication stress, and activation of the ATR pathway in AML cells. PIWIL4 depletion potentiates sensitivity to pharmacological inhibition of the ATR pathway and creates a pharmacologically actionable dependency in AML cells.

Immunophenotypical profiling of myeloid neoplasms with erythroid predominance using mass cytometry (CyTOF).

Acute erythroid leukemia (AEL) is a disease continuum between Myelodysplastic syndrome (MDS) and Acute myeloid leukemia (AML) with the cellular hallmark of uncontrolled proliferation and impaired differentiation of erythroid progenitor cells. First described by Giovanni di Guglielmo in 1917 AEL accounts for less than 5% of all de novo AML cases. There have been efforts to characterize AEL at a molecular level, describing recurrent alterations in TP53, NPM1 and FLT3 genes. A genomic analysis of AEL cases confirmed its complexity. Despite these advances, the biology underlying erythroid proliferations remains unclear and the prognosis is dismal with a median survival of only 3 months for pure erythroid leukemia (PEL). Marker combinations suitable for the identification and characterization of leukemic stem cell (LSC) candidates, monitoring measurable residual disease (MRD) during chemotherapy treatment and the development of innovative targeted therapies are missing. Here, we developed a mass cytometry panel for an in-depth characterization of erythroid and myeloid blast cell populations from human AEL bone marrow samples in comparison to other AML subtypes and healthy counterparts. A total of 8 AEL samples were analyzed and compared to 28 AML samples from different molecular subtypes, healthy bone marrow counterparts (n = 5) and umbilical cord blood (n = 6) using high-dimensional mass cytometry. Identification of erythroid and myeloid blast populations in high-dimensional mass cytometry data enabled a refined view on erythroblast differentiation stages present in AEL erythroid blasts and revealed immunophenotypical profiles specific to AEL. Profiling of phenotypic LSCs revealed aberrant erythroid marker expression in the CD34+ CD38- stem cell compartment. In addition, the identification of novel candidate surface marker combinations and aberrancies might enhance clinical diagnostics of AEL. We present a high-parameter mass cytometry approach feasible for immunophenotypical analysis of blast and stem cell populations in myeloid neoplasms with erythroid predominance laying the foundation for more precise experimental approaches in the future.

TET3 promotes AML growth and epigenetically regulates glucose metabolism and leukemic stem cell associated pathways.

Acute myeloid leukemia (AML) is considered a poor prognosis malignancy where patients exhibit altered glucose metabolism and stem cell signatures that contribute to AML growth and maintenance. Here, we report that the epigenetic factor, Ten-Eleven Translocation 3 (TET3) dioxygenase is overexpressed in AML patients and functionally validated human leukemic stem cells (LSCs), is required for leukemic growth by virtue of its regulation of glucose metabolism in AML cells. In human AML cells, TET3 maintains 5-hydroxymethylcytosine (5hmC) epigenetic marks and expression of early myeloid progenitor program, critical glucose metabolism and STAT5A signaling pathway genes, which also positively correlate with TET3 expression in AML patients. Consequently, TET3 depletion impedes hexokinase activity and L-Lactate production in AML cells. Conversely, overexpression of TET3 in healthy human hematopoietic stem progenitors (HSPCs) upregulates the expression of glucose metabolism, STAT5A signaling and AML associated genes, and impairs normal HSPC lineage differentiation in vitro. Finally, TET3 depletion renders AML cells highly sensitive to blockage of the TET3 downstream pathways glycolysis and STAT5 signaling via the combination of 2-Deoxy-D-glucose and STAT5 inhibitor which preferentially targets AML cells but spares healthy CD34+ HSPCs.

Somatostatin receptor mediated targeting of acute myeloid leukemia by photodynamic metal complexes for light induced apoptosis.

Acute myeloid leukemia (AML) is characterized by relapse and treatment resistance in a major fraction of patients, underlining the need of innovative AML targeting therapies. Here we analysed the therapeutic potential of an innovative biohybrid consisting of the tumor-associated peptide somatostatin and the photosensitizer ruthenium in AML cell lines and primary AML patient samples. Selective toxicity was analyzed by using CD34 enriched cord blood cells as control. Treatment of OCI AML3, HL60 and THP1 resulted in a 92, and 99 and 97% decrease in clonogenic growth compared to the controls. Primary AML cells demonstrated a major response with a 74 to 99% reduction in clonogenicity in 5 of 6 patient samples. In contrast, treatment of CD34+ CB cells resulted in substantially less reduction in colony numbers. Subcellular localization assays of RU-SST in OCI-AML3 cells confirmed strong co-localization of RU-SST in the lysosomes compared to the other cellular organelles. Our data demonstrate that conjugation of a Ruthenium complex with somatostatin is efficiently eradicating LSC candidates of patients with AML. This indicates that receptor mediated lysosomal accumulation of photodynamic metal complexes is a highly attractive approach for targeting AML cells.

Glutathione peroxidase 4 and vitamin E control reticulocyte maturation, stress erythropoiesis and iron homeostasis.

Glutathione peroxidase 4 (GPX4) is unique as it is the only enzyme that can prevent detrimental lipid peroxidation in vivo by reducing lipid peroxides to the respective alcohols thereby stabilizing oxidation products of unsaturated fatty acids. During reticulocyte maturation, lipid peroxidation mediated by 15-lipoxygenase in humans and rabbits and by 12/15-lipoxygenase (ALOX15) in mice was considered the initiating event for the elimination of mitochondria but is now known to occur through mitophagy. Yet, genetic ablation of the Alox15 gene in mice failed to provide evidence for this hypothesis. We designed a different genetic approach to tackle this open conundrum. Since either other lipoxygenases or non-enzymatic autooxidative mechanisms may compensate for the loss of Alox15, we asked whether ablation of Gpx4 in the hematopoietic system would result in the perturbation of reticulocyte maturation. Quantitative assessment of erythropoiesis indices in the blood, bone marrow (BM) and spleen of chimeric mice with Gpx4 ablated in hematopoietic cells revealed anemia with an increase in the fraction of erythroid precursor cells and reticulocytes. Additional dietary vitamin E depletion strongly aggravated the anemic phenotype. Despite strong extramedullary erythropoiesis reticulocytes failed to mature and accumulated large autophagosomes with engulfed mitochondria. Gpx4-deficiency in hematopoietic cells led to systemic hepatic iron overload and simultaneous severe iron demand in the erythroid system. Despite extremely high erythropoietin and erythroferrone levels in the plasma, hepcidin expression remained unchanged. Conclusively, perturbed reticulocyte maturation in response to Gpx4 loss in hematopoietic cells thus causes ineffective erythropoiesis, a phenotype partially masked by dietary vitamin E supplementation.

The ParaHox gene Cdx4 induces acute erythroid leukemia in mice.

Acute erythroid leukemia (AEL) is a rare and aggressive form of acute leukemia, the biology of which remains poorly understood. Here we demonstrate that the ParaHox gene CDX4 is expressed in patients with acute erythroid leukemia, and that aberrant expression of Cdx4 induced homogenously a transplantable acute erythroid leukemia in mice. Gene expression analyses demonstrated upregulation of genes involved in stemness and leukemogenesis, with parallel downregulation of target genes of Gata1 and Gata2 responsible for erythroid differentiation. Cdx4 induced a proteomic profile that overlapped with a cluster of proteins previously defined to represent the most primitive human erythroid progenitors. Whole-exome sequencing of diseased mice identified recurrent mutations significantly enriched for transcription factors involved in erythroid lineage specification, as well as TP53 target genes partly identical to the ones reported in patients with AEL. In summary, our data indicate that Cdx4 is able to induce stemness and inhibit terminal erythroid differentiation, leading to the development of AEL in association with co-occurring mutations.

Mitochondria Targeted Protein-Ruthenium Photosensitizer for Efficient Photodynamic Applications.

Organelle-targeted photosensitization represents a promising approach in photodynamic therapy where the design of the active photosensitizer (PS) is very crucial. In this work, we developed a macromolecular PS with multiple copies of mitochondria-targeting groups and ruthenium complexes that displays highest phototoxicity toward several cancerous cell lines. In particular, enhanced anticancer activity was demonstrated in acute myeloid leukemia cell lines, where significant impairment of proliferation and clonogenicity occurs. Finally, attractive two-photon absorbing properties further underlined the great significance of this PS for mitochondria targeted PDT applications in deep tissue cancer therapy.

Controlled stem cell amplification by HOXB4 depends on its unique proline-rich region near the N terminus.

There is high interest in understanding the mechanisms that drive self-renewal of stem cells. HOXB4 is one of the few transcription factors that can amplify long-term repopulating hematopoietic stem cells in a controlled way. Here we show in mice that this characteristic of HOXB4 depends on a proline-rich sequence near the N terminus, which is unique among HOX genes and highly conserved in higher mammals. Deletion of this domain substantially enhanced the oncogenicity of HOXB4, inducing acute leukemia in mice. Conversely, insertion of the domain into Hoxa9 impaired leukemogenicity of this homeobox gene. These results indicate that proline-rich stretches attenuate the potential of stem cell active homeobox genes to acquire oncogenic properties.

VENTX induces expansion of primitive erythroid cells and contributes to the development of acute myeloid leukemia in mice.

Homeobox genes are key regulators in normal and malignant hematopoiesis. The human Vent-like homeobox gene VENTX, a putative homolog of the Xenopus laevis Xvent-2 gene, was shown to be highly expressed in normal myeloid cells and in patients with acute myeloid leukemia. We now demonstrate that constitutive expression of VENTX suppresses expression of genes responsible for terminal erythroid differentiation in normal CD34+ stem and progenitor cells. Transplantation of bone marrow progenitor cells retrovirally engineered to express VENTX caused massive expansion of primitive erythroid cells and partly acute erythroleukemia in transplanted mice. The leukemogenic potential of VENTX was confirmed in the AML1-ETO transplantation model, as in contrast to AML1-ETO alone co-expression of AML1-ETO and VENTX induced acute myeloid leukemia, partly expressing erythroid markers, in all transplanted mice. VENTX was highly expressed in patients with primary human erythroleukemias and knockdown of VENTX in the erythroleukemic HEL cell line significantly blocked cell growth. In summary, these data indicate that VENTX is able to perturb erythroid differentiation and to contribute to myeloid leukemogenesis when co-expressed with appropriate AML oncogenes and point to its potential significance as a novel therapeutic target in AML.

MEIS2 Is an Oncogenic Partner in AML1-ETO-Positive AML.

Homeobox genes are known to be key factors in leukemogenesis. Although the TALE family homeodomain factor Meis1 has been linked to malignancy, a role for MEIS2 is less clear. Here, we demonstrate that MEIS2 is expressed at high levels in patients with AML1-ETO-positive acute myeloid leukemia and that growth of AML1-ETO-positive leukemia depends on MEIS2 expression. In mice, MEIS2 collaborates with AML1-ETO to induce acute myeloid leukemia. MEIS2 binds strongly to the Runt domain of AML1-ETO, indicating a direct interaction between these transcription factors. High expression of MEIS2 impairs repressive DNA binding of AML1-ETO, inducing increased expression of genes such as the druggable proto-oncogene YES1. Collectively, these data describe a pivotal role for MEIS2 in AML1-ETO-induced leukemia.