Protective mitochondrial transfer from bone marrow stromal cells to acute myeloid leukemic cells during chemotherapy.

Here we demonstrate that in a niche-like coculture system, cells from both primary and cultured acute myeloid leukemia (AML) sources take up functional mitochondria from murine or human bone marrow stromal cells. Using different molecular and imaging approaches, we show that AML cells can increase their mitochondrial mass up to 14%. After coculture, recipient AML cells showed a 1.5-fold increase in mitochondrial adenosine triphosphate production and were less prone to mitochondrial depolarization after chemotherapy, displaying a higher survival. This unidirectional transfer enhanced by some chemotherapeutic agents required cell-cell contacts and proceeded through an endocytic pathway. Transfer was greater in AML blasts compared with normal cord blood CD34(+) cells. Finally, we demonstrate that mitochondrial transfer was observed in vivo in an NSG immunodeficient mouse xenograft model and also occurred in human leukemia initiating cells and progenitors. As mitochondrial transfer provides a clear survival advantage following chemotherapy and a higher leukemic long-term culture initiating cell potential, targeting mitochondrial transfer could represent a future therapeutic target for AML treatment.

Dexamethasone in hyperleukocytic acute myeloid leukemia.

Patients with acute myeloid leukemia and a high white blood cell count are at increased risk of early death and relapse. Because mediators of inflammation contribute to leukostasis and chemoresistance, dexamethasone added to chemotherapy could improve outcomes. This retrospective study evaluated the impact of adding or not adding dexamethasone to chemotherapy in a cohort of 160 patients with at least 50×109 white blood cells. In silico studies, primary samples, leukemic cell lines, and xenograft mouse models were used to explore the antileukemic activity of dexamethasone. There was no difference with respect to induction death rate, response, and infections between the 60 patients in the dexamethasone group and the 100 patients in the no dexamethasone group. Multivariate analysis showed that dexamethasone was significantly associated with improved relapse incidence (adjusted sub-HR: 0.30; 95% CI: 0.14-0.62; P=0.001), disease-free survival (adjusted HR: 0.50; 95% CI: 0.29-0.84; P=0.010), event-free survival (adjusted HR: 0.35; 95% CI: 0.21-0.58; P<0.001), and overall survival (adjusted HR: 0.41; 95% CI: 0.22-0.79; P=0.007). In a co-culture system, dexamethasone reduced the frequency of leukemic long-term culture initiating cells by 38% and enhanced the cytotoxicity of doxorubicin and cytarabine. In a patient-derived xenograft model treated with cytarabine, chemoresistant cells were enriched in genes of the inflammatory response modulated by dexamethasone. Dexamethasone also demonstrated antileukemic activity in NPM1-mutated samples. Dexamethasone may improve the outcome of acute myeloid leukemia patients receiving intensive chemotherapy. This effect could be due to the modulation of inflammatory chemoresistance pathways and to a specific activity in acute myeloid leukemia with NPM1 mutation.

Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation.

Acute myeloid leukemia (AML) induces bone marrow (BM) failure in patients, predisposing them to life-threatening infections and bleeding. The mechanism by which AML mediates this complication is unknown but one widely accepted explanation is that AML depletes the BM of hematopoietic stem cells (HSCs) through displacement. We sought to investigate how AML affects hematopoiesis by quantifying residual normal hematopoietic subpopulations in the BM of immunodeficient mice transplanted with human AML cells with a range of genetic lesions. The numbers of normal mouse HSCs were preserved whereas normal progenitors and other downstream hematopoietic cells were reduced following transplantation of primary AMLs, findings consistent with a differentiation block at the HSC-progenitor transition, rather than displacement. Once removed from the leukemic environment, residual normal hematopoietic cells differentiated normally and outcompeted steady-state hematopoietic cells, indicating that this effect is reversible. We confirmed the clinical significance of this by ex vivo analysis of normal hematopoietic subpopulations from BM of 16 patients with AML. This analysis demonstrated that the numbers of normal CD34(+)CD38(-) stem-progenitor cells were similar in the BM of AML patients and controls, whereas normal CD34(+)CD38(+) progenitors were reduced. Residual normal CD34(+) cells from patients with AML were enriched in long-term culture, initiating cells and repopulating cells compared with controls. In conclusion the data do not support the idea that BM failure in AML is due to HSC depletion. Rather, AML inhibits production of downstream hematopoietic cells by impeding differentiation at the HSC-progenitor transition.

Automated cell type annotation and exploration of single-cell signaling dynamics using mass cytometry.

Mass cytometry by time-of-flight (CyTOF) is an emerging technology allowing for in-depth characterization of cellular heterogeneity in cancer and other diseases. Unfortunately, high-dimensional analyses of CyTOF data remain quite demanding. Here, we deploy a bioinformatics framework that tackles two fundamental problems in CyTOF analyses namely (1) automated annotation of cell populations guided by a reference dataset and (2) systematic utilization of single-cell data for effective patient stratification. By applying this framework on several publicly available datasets, we demonstrate that the Scaffold approach achieves good trade-off between sensitivity and specificity for automated cell type annotation. Additionally, a case study focusing on a cohort of 43 leukemia patients reported salient interactions between signaling proteins that are sufficient to predict short-term survival at time of diagnosis using the XGBoost algorithm. Our work introduces an automated and versatile analysis framework for CyTOF data with many applications in future precision medicine projects.

Oxidative Phosphorylation Fueled by Fatty Acid Oxidation Sensitizes Leukemic Stem Cells to Cold.

Dependency on mitochondrial oxidative phosphorylation (OxPhos) is a potential weakness for leukemic stem cells (LSC) that can be exploited for therapeutic purposes. Fatty acid oxidation (FAO) is a crucial OxPhos-fueling catabolic pathway for some acute myeloid leukemia (AML) cells, particularly chemotherapy-resistant AML cells. Here, we identified cold sensitivity at 4°C (cold killing challenge; CKC4), commonly used for sample storage, as a novel vulnerability that selectively kills AML LSCs with active FAO-supported OxPhos while sparing normal hematopoietic stem cells. Cell death of OxPhos-positive leukemic cells was induced by membrane permeabilization at 4°C; by sharp contrast, leukemic cells relying on glycolysis were resistant. Forcing glycolytic cells to activate OxPhos metabolism sensitized them to CKC4. Lipidomic and proteomic analyses showed that OxPhos shapes the composition of the plasma membrane and introduces variation of 22 lipid subfamilies between cold-sensitive and cold-resistant cells. Together, these findings indicate that steady-state energy metabolism at body temperature predetermines the sensitivity of AML LSCs to cold temperature, suggesting that cold sensitivity could be a potential OxPhos biomarker. These results could have important implications for designing experiments for AML research to avoid cell storage at 4°C. SIGNIFICANCE: Mitochondrial metabolism fueled by FAO alters the membrane composition and introduces membrane fragility upon cold exposure in OxPhos-driven AML and in LSCs. See related commentary by Jones, p. 2441.

"Microenvironmental contaminations" induced by fluorescent lipophilic dyes used for noninvasive in vitro and in vivo cell tracking.

Determining how normal and leukemic stem cells behave in vivo, in a dynamic and noninvasive way, remains a major challenge. Most optical tracking technologies rely on the use of fluorescent or bioluminescent reporter genes, which need to be stably expressed in the cells of interest. Because gene transfer in primary leukemia samples represents a major risk to impair their capability to engraft in a xenogenic context, we evaluated the possibility to use gene transfer-free labeling technologies. The lipophilic dye 3,3,3',3' tetramethylindotricarbocyanine iodide (DiR) was selected among 4 near-infrared (NIR) staining technologies. Unfortunately we report here a massive transfer of the dye occurring toward the neighbor cells both in vivo and in vitro. We further demonstrate that all lipophilic dyes tested in this study (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine perchlorate [DiI], DiD, DiR, and PKH26) can give rise to microenvironmental contamination, including when used in suboptimal concentration, after extensive washing procedures and in the absence of phagocytosis or marked cell death. This was observed from all cell types tested. Eventually, we show that this microenvironmental contamination is mediated by both direct cell-cell contacts and diffusible microparticles. We conclude that tracking of labeled cells using non-genetically encoded markers should always be accompanied by drastic cross validation using multimodality approaches.

Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34(-) fraction.

Leukemia-initiating cells (LICs) in acute myeloid leukemia (AML) are believed to be restricted to the CD34(+) fraction. However, one of the most frequently mutated genes in AML is nucleophosmin (NPM), and this is associated with low CD34 expression. We, therefore, investigated whether NPM-mutated AMLs have LICs restricted to the CD34(+) fraction. We transplanted sorted fractions of primary NPM-mutated AML into immunodeficient mice to establish which fractions initiate leukemia. Approximately one-half of cases had LICs exclusively within the CD34(-) fraction, whereas the CD34(+) fraction contained normal multilineage hematopoietic repopulating cells. Most of the remaining cases had LICs in both CD34(+) and CD34(-) fractions. When samples were sorted based on CD34 and CD38 expression, multiple fractions initiated leukemia in primary and secondary recipients. The data indicate that the phenotype of LICs is more heterogeneous than previously realized and can vary even within a single sample. This feature of LICs may make them particularly difficult to eradicate using therapies targeted against surface antigens.

Aerodynamically assisted bio-jetting of hematopoietic stem cells.

The research reported in this communication demonstrates the emerging direct cell handling technology now widely referred to as aerodynamically assisted bio-jetting. This is a non-electric field driven approach which directly competes with bio-electrosprays. The technology in these investigations has been explored for the direct handling of live murine primary hematopoietic stem cells. The viability studies demonstrate the complete inertness of this technology for handling such cells for a wide range of applications in both basic biology and clinical medicine. Interestingly these studies pave the way for this technology to undergo development as a flow cell for utility as a sheathless cell most useful in flow cytometry.

The PIP4K2 inhibitor THZ-P1-2 exhibits antileukemia activity by disruption of mitochondrial homeostasis and autophagy.

The treatment of acute leukemia is challenging because of the genetic heterogeneity between and within patients. Leukemic stem cells (LSCs) are relatively drug-resistant and frequently relapse. Their plasticity and capacity to adapt to extracellular stress, in which mitochondrial metabolism and autophagy play important roles, further complicates treatment. Genetic models of phosphatidylinositol-5-phosphate 4-kinase type 2 protein (PIP4K2s) inhibition have demonstrated the relevance of these enzymes in mitochondrial homeostasis and autophagic flux. Here, we uncovered the cellular and molecular effects of THZ-P1-2, a pan-inhibitor of PIP4K2s, in acute leukemia cells. THZ-P1-2 reduced cell viability and induced DNA damage, apoptosis, loss of mitochondrial membrane potential, and the accumulation of acidic vesicular organelles. Protein expression analysis revealed that THZ-P1-2 impaired autophagic flux. In addition, THZ-P1-2 induced cell differentiation and showed synergistic effects with venetoclax. In primary leukemia cells, LC-MS/MS-based proteome analysis revealed that sensitivity to THZ-P1-2 is associated with mitochondrial metabolism, cell cycle, cell-of-origin (hematopoietic stem cell and myeloid progenitor), and the TP53 pathway. The minimal effects of THZ-P1-2 observed in healthy CD34+ cells suggest a favorable therapeutic window. Our study provides insights into the pharmacological inhibition of PIP4K2s targeting mitochondrial homeostasis and autophagy, shedding light on a new class of drugs for acute leukemia.

Acute myeloid leukemia xenograft success prediction: Saving time.

Xenograft assay allows functional analysis of leukemia-initiating cells of acute myeloid leukemia primary samples. However, 40% of samples derived from patients with better outcomes fail to engraft in immunodeficient mouse recipients when conventional protocols are followed. At diagnosis, the engraftment of intermediate-risk group samples cannot be anticipated. In this study, we decided to further explore the reasons for xenograft success and failure. No differences in extracellular phenotype, apoptosis, or cell cycle profile could distinguish samples that engraft (engrafter [E]) from samples that do not engraft (nonengrafter [NE]) in NSG mice. In addition, ex vivo long-term culture assay revealed, after 5 weeks, a lower content of leukemic-LTC-initiating cells in the NE samples associated with a lower expansion rate capacity. One-week co-cultures with mesenchymal or osteoblastic or endothelial cells did not influence the proliferation rate, suggesting that E and NE samples are genuinely rapidly or slowly expanding independent of external cue. Engraftment success for some NE samples was consistently observed in recipient mice analyzed 6 months later than the conventional 3-month period. Eventually we implemented a flow cytometry-based assay, which allowed us to predict, in 1 week, the fast or delayed engraftment potential of a noncharacterized acute myeloid leukemia sample. This approach will be especially useful in selecting intermediate-risk-group patient samples and restricting the experimental duration to a 3-month period and, eventually, in reducing the number of animals and the cost and effort of unnecessary xenograft failures.

Intercellular mitochondria trafficking highlighting the dual role of mesenchymal stem cells as both sensors and rescuers of tissue injury.

Mitochondria are crucial organelles that not only regulate the energy metabolism, but also the survival and fate of eukaryotic cells. Mitochondria were recently discovered to be able to translocate from one cell to the other. This phenomenon was observed in vitro and in vivo, both in physiological and pathophysiological conditions including tissue injury and cancer. Mitochondria trafficking was found to exert prominent biological functions. In particular, several studies pointed out that this process governs some of the therapeutic effects of mesenchymal stem cells (MSCs). In this review, we give an overview of the current knowledge on MSC-dependent intercellular mitochondria trafficking and further discuss the recent findings on the intercellular mitochondria transfer between differentiated and mesenchymal stem cells, their biological significance and the mechanisms underlying this process.

Mitochondrial Transfer in the Leukemia Microenvironment.

The bone marrow microenvironment (BMME) is a complex ecosystem that instructs and protects hematopoietic stem cells (HSCs) and their malignant counterparts, the leukemia-initiating cells (LICs). Within the physical and functional crosstalk that takes place between HSCs, LICs, and the BMME, the transfer of organelles and of mitochondria in particular is an important new intercellular communication mode in addition to adhesion molecules, tunneling nanotubes (TNTs), and the paracrine secretion of cytokines, (onco)metabolites, and extracellular vesicles (EVs). In this review we discuss the functional roles of mitochondrial transfer between BMME and leukemic cells, and give insights into this new mechanism of drug resistance whose understanding will open the way to innovative anticancer adjuvant treatments.

Iron chelation: an adjuvant therapy to target metabolism, growth and survival of murine PTEN-deficient T lymphoma and human T lymphoblastic leukemia/lymphoma.

Iron is an essential nutrient, acting as a catalyst for metabolic reactions that are fundamental to cell survival and proliferation. Iron complexed to transferrin is delivered to the metabolism after endocytosis via the CD71 surface receptor. We found that transformed cells from a murine PTEN-deficient T-cell lymphoma model and from T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/T-LL) cell lines overexpress CD71. As a consequence, the cells developed an addiction toward iron whose chelation by deferoxamine (DFO) dramatically affected their survival to induce apoptosis. Interestingly, DFO displayed synergistic activity with three ALL-specific drugs: dexamethasone, doxorubicin, and L-asparaginase. DFO appeared to act through a reactive oxygen species-dependent DNA damage response and potentiated the action of an inhibitor of the PARP pathway of DNA repair. Our results demonstrate that targeting iron metabolism could be an interesting adjuvant therapy for acute lymphoblastic leukemia.

Chemotherapy-Resistant Human Acute Myeloid Leukemia Cells Are Not Enriched for Leukemic Stem Cells but Require Oxidative Metabolism.

Chemotherapy-resistant human acute myeloid leukemia (AML) cells are thought to be enriched in quiescent immature leukemic stem cells (LSC). To validate this hypothesis in vivo, we developed a clinically relevant chemotherapeutic approach treating patient-derived xenografts (PDX) with cytarabine (AraC). AraC residual AML cells are enriched in neither immature, quiescent cells nor LSCs. Strikingly, AraC-resistant preexisting and persisting cells displayed high levels of reactive oxygen species, showed increased mitochondrial mass, and retained active polarized mitochondria, consistent with a high oxidative phosphorylation (OXPHOS) status. AraC residual cells exhibited increased fatty-acid oxidation, upregulated CD36 expression, and a high OXPHOS gene signature predictive for treatment response in PDX and patients with AML. High OXPHOS but not low OXPHOS human AML cell lines were chemoresistant in vivo. Targeting mitochondrial protein synthesis, electron transfer, or fatty-acid oxidation induced an energetic shift toward low OXPHOS and markedly enhanced antileukemic effects of AraC. Together, this study demonstrates that essential mitochondrial functions contribute to AraC resistance in AML and are a robust hallmark of AraC sensitivity and a promising therapeutic avenue to treat AML residual disease.Significance: AraC-resistant AML cells exhibit metabolic features and gene signatures consistent with a high OXPHOS status. In these cells, targeting mitochondrial metabolism through the CD36-FAO-OXPHOS axis induces an energetic shift toward low OXPHOS and strongly enhanced antileukemic effects of AraC, offering a promising avenue to design new therapeutic strategies and fight AraC resistance in AML. Cancer Discov; 7(7); 716-35. ©2017 AACR.See related commentary by Schimmer, p. 670This article is highlighted in the In This Issue feature, p. 653.

Frequency and Dynamics of Leukemia-Initiating Cells during Short-term Ex Vivo Culture Informs Outcomes in Acute Myeloid Leukemia Patients.

Acute myeloid leukemia (AML) is sustained by a subpopulation of rare leukemia-initiating cells (LIC) detected in the xenograft assay by their capacity to self-renew and to generate non-LICs in vivo The xenotransplantation model captures functional properties of LICs that have clinical prognostic value. However, the long duration of this in vivo assay has hampered its use as a prognostic tool. Here, we show, using an ex vivo coculture system, that intermediate and poor risk AML patient samples at diagnosis have a 5 to 7 times higher frequency of leukemic long-term culture-initiating cells (L-LTC-IC) compared with the good risk group. We defined a fluorescence dilution factor (FDF) parameter that monitors sample proliferation over 1 week and established a strong correlation of this parameter with the L-LTC-IC frequency. A higher FDF was found for poor prognostic AMLs or for samples capable of engrafting NSG mice compared with good risk AMLs or nonengrafters. Importantly, FDF could classify normal karyotype intermediate risk patients into two groups with a significant difference in their overall survival, thus making this nongenetic and non-in vivo approach a new clinically relevant tool for better diagnosis of AML patients. Cancer Res; 76(8); 2082-6. ©2016 AACR.

BCL-B (BCL2L10) is overexpressed in patients suffering from multiple myeloma (MM) and drives an MM-like disease in transgenic mice.

Multiple myeloma (MM) evolves from a premalignant condition known as monoclonal gammopathy of undetermined significance (MGUS). However, the factors underlying the malignant transformation of plasmocytes in MM are not fully characterized. We report here that Eµ-directed expression of the antiapoptotic Bcl-B protein in mice drives an MM phenotype that reproduces accurately the human disease. Indeed, with age, Eµ-bcl-b transgenic mice develop the characteristic features of human MM, including bone malignant plasma cell infiltration, a monoclonal immunoglobulin peak, immunoglobulin deposit in renal tubules, and highly characteristic bone lytic lesions. In addition, the tumors are serially transplantable in irradiated wild-type mice, underlying the tumoral origin of the disease. Eµ-bcl-b plasmocytes show increased expression of a panel of genes known to be dysregulated in human MM pathogenesis. Treatment of Eµ-bcl-b mice with drugs currently used to treat patients such as melphalan and VELCADE efficiently kills malignant plasmocytes in vivo. Finally, we find that Bcl-B is overexpressed in plasmocytes from MM patients but neither in MGUS patients nor in healthy individuals, suggesting that Bcl-B may drive MM. These findings suggest that Bcl-B could be an important factor in MM disease and pinpoint Eµ-bcl-b mice as a pertinent model to validate new therapies in MM.

AS602868, a pharmacological inhibitor of IKK2, reveals the apoptotic potential of TNF-alpha in Jurkat leukemic cells.

NF-kappaB transcription factors promote survival in numerous cell types via induction of antiapoptotic genes. Pharmacological blockade of the IKK2 kinase with AS602868, a specific inhibitor that competes with ATP binding, prevented TNF-alpha-induced NF-kappaB activation in Jurkat leukemic T cells. While TNF-alpha by itself had no effect on Jurkat survival, the addition of AS602868 induced cell death, visualized by DNA fragmentation and sub-G1 analysis. A disruption of the mitochondrial potential followed by activation of caspases 9 and 3 was observed in cells treated by the combination TNF-alpha+AS602868. Quantitative real-time PCR demonstrated that AS602868 prevented TNF-alpha induction of the antiapoptotic genes coding for c-IAP-2, Bclx, Bfl-1/A1 and Traf-1. The use of a specific IKK2 inhibitor appears, therefore, as an interesting pharmaceutical strategy to increase the cell's sensitivity towards apoptotic effectors.

APOBEC3A is implicated in a novel class of G-to-A mRNA editing in WT1 transcripts.

Classic deamination mRNA changes, including cytidine to uridine (C-to-U) and adenosine to inosine (A-to-I), are important exceptions to the central dogma and lead to significant alterations in gene transcripts and products. Although there are a few reports of non-classic mRNA alterations, as yet there is no molecular explanation for these alternative changes. Wilms Tumor 1 (WT1) mutations and variants are implicated in several diseases, including Wilms tumor and acute myeloid leukemia (AML). We observed two alternative G-to-A changes, namely c.1303G>A and c.1586G>A in cDNA clones and found them to be recurrent in a series of 21 umbilical cord blood mononuclear cell (CBMC) samples studied. Two less conserved U-to-C changes were also observed. These alternative changes were found to be significantly higher in non-progenitor as compared to progenitor CBMCs, while they were found to be absent in a series of AML samples studied, indicating they are targeted, cell type-specific mRNA editing modifications. Since APOBEC/ADAR family members are implicated in RNA/DNA editing, we screened them by RNA-interference (RNAi) for WT1-mRNA changes and observed near complete reversal of WT1 c.1303G>A alteration upon APOBEC3A (A3A) knockdown. The role of A3A in mediating this change was confirmed by A3A overexpression in Fujioka cells, which led to a significant increase in WT1 c.1303G>A mRNA editing. Non-progenitor CBMCs showed correspondingly higher levels of A3A-mRNA and protein as compared to the progenitor ones. To our knowledge, this is the first report of mRNA modifying activity for an APOBEC3 protein and implicates A3A in a novel G-to-A form of editing. These findings open the way to further investigations into the mechanisms of other potential mRNA changes, which will help to redefine the RNA editing paradigm in both health and disease.

A niche-like culture system allowing the maintenance of primary human acute myeloid leukemia-initiating cells: a new tool to decipher their chemoresistance and self-renewal mechanisms.

Acute myeloid leukemia-initiating cells (LICs) are responsible for the emergence of leukemia and relapse after chemotherapy. Despite their identification more than 15 years ago, our understanding of the mechanisms responsible for their self-renewal activity and their chemoresistance remains poor. The slow progress in this area is partly due to the difficulty of studying these cells ex vivo. Indeed, current studies are reliant on xenotransplantation assays in immunodeficient mice. In this paper, we report that by modeling key elements of the bone marrow niche using different stromal feeder layers and hypoxic culture conditions, we can maintain LICs over at least 3 weeks and support their self-renewal properties demonstrated through primary and secondary successful xenograft. We provide a proof of principle that this niche-like culture system can be used to study LIC chemoresistance following in vitro cytarabine treatment similarly to the xenograft chemotherapy model. We found that although LICs are believed to be more chemoresistant than non-LICs, functionally defined LICs are not enriched after cytarabine treatment, and heterogeneity in their resistance to treatment can be seen between patients and even within the same patient. We present a culture system that can be used as an in vitro surrogate for xenotransplantation and that has the potential to dramatically increase the throughput of the investigation of LICs. This would further provide the means by which to identify and target the functionality of the different signaling pathways involved in the maintenance and resistance of LICs to improve acute myeloid leukemia treatments.