PP2A is a therapeutically targetable driver of cell fate decisions via a c-Myc/p21 axis in human and murine acute myeloid leukemia.

Dysregulated cellular differentiation is a hallmark of acute leukemogenesis. Phosphatases are widely suppressed in cancers but have not been traditionally associated with differentiation. In this study, we found that the silencing of protein phosphatase 2A (PP2A) directly blocks differentiation in acute myeloid leukemia (AML). Gene expression and mass cytometric profiling revealed that PP2A activation modulates cell cycle and transcriptional regulators that program terminal myeloid differentiation. Using a novel pharmacological agent, OSU-2S, in parallel with genetic approaches, we discovered that PP2A enforced c-Myc and p21 dependent terminal differentiation, proliferation arrest, and apoptosis in AML. Finally, we demonstrated that PP2A activation decreased leukemia-initiating stem cells, increased leukemic blast maturation, and improved overall survival in murine Tet2-/-Flt3ITD/WT and human cell-line derived xenograft AML models in vivo. Our findings identify the PP2A/c-Myc/p21 axis as a critical regulator of the differentiation/proliferation switch in AML that can be therapeutically targeted in malignancies with dysregulated maturation fate.

Alternative methods of viability determination in single cell mass cytometry.

The identification and discrimination of viable cells is important to understand how experimental variables may influence biochemical processes such as cell metabolism, cell cycle, and signaling pathways. Cisplatin is commonly used as a viability stain in mass cytometry studies, however, recent work by Mei et al. has demonstrated that cisplatin can also be used to label antibodies, complicating the simultaneous use of the platinum measurement channels for both antibody and viability staining. This study demonstrates that other metal salts (hafnium chloride, niobium chloride, and zirconium chloride) can serve as substitutes for cisplatin in viability staining. These stains yield similar fractions of live and dead cells and stain the same dead cells in parallel high parameter analyses. In addition, this study demonstrates how a variety of protein antigen viability markers (pRb, Ki-67, Histone H1, cleaved PARP, and GAPDH) can be used to discriminate live and dead cell populations, without the need for a separate viability staining step. As few as two of these protein antigen viability markers can help identify live and dead cell populations in fixed samples and can identify the same viable cells in high dimensional analyses with or without use of viability stain information. This study demonstrates several alternative approaches to mass cytometry viability assessment that can facilitate use of platinum isotopes for antibody staining and enables identification of live and dead cell populations in samples for which a separate viability stain is not practical.

Effect of storage time and temperature on cell cycle analysis by mass cytometry.

Cell cycle analysis is a recognized and important application of flow cytometry and, more recently, mass cytometry (MCM). Both technologies have been utilized for analysis of the cell cycle state of ex vivo samples from patients with hematologic malignancies. Clinical samples are frequently stored for hours at room temperature or cryogenically frozen before processing and analysis; however, how these processing methods alter cell cycle state is not well described. To understand how storage time and temperature affect the analysis of cell cycle distribution by MCM, two leukemia cell lines, HL-60 and MOLM13, and primary human cells from three human bone marrow aspirates were stored and frozen under a variety of conditions that are likely to be encountered in a clinical setting. Our findings indicate that short delays in sample processing (less than 1 h), have little to no effect on cell cycle distribution, while longer delays or cryopreservation cause significant disruptions to the cell cycle fraction characterized by consistent reductions in IdU incorporation and variable alterations in other cell cycle phases. Analysis of the recovery of cryopreserved leukemia cell lines and marrow cells demonstrated that cell cycle alterations persist for at least 48 h after thawing. Our findings demonstrate that accurate cell cycle analysis requires that samples be processed rapidly after collection, and that cryopreservation significantly alters cell cycle fractions. Measurement of IdU incorporation was the most sensitive to both delays in processing and cryopreservation, while estimation of the total cycling cell fraction using Ki-67 or phosphorylated retinoblastoma protein were least altered by the conditions tested. These findings provide guidance for the ideal approach to collection of samples for cell cycle analysis and can aid interpretation of cell cycle data from samples that cannot be collected under ideal circumstances. © 2018 International Society for Advancement of Cytometry.

Increased hypoxia-inducible factor-1α in striated muscle of tumor-bearing mice.

Cancer cachexia is a progressive wasting disease resulting in significant effects on the quality of life and high mortality. Most studies on cancer cachexia have focused on skeletal muscle; however, the heart is now recognized as a major site of cachexia-related effects. To elucidate possible mechanisms, a proteomic study was performed on the left ventricles of colon-26 (C26) adenocarcinoma tumor-bearing mice. The results revealed several changes in proteins involved in metabolism. An integrated pathway analysis of the results revealed a common mediator in hypoxia-inducible factor-1α (HIF-1α). Work by other laboratories has shown that extensive metabolic restructuring in the C26 mouse model causes changes in gene expression that may be affected directly by HIF-1α, such as glucose metabolic genes. M-mode echocardiography showed progressive decline in heart function by day 19, exhibited by significantly decreased ejection fraction and fractional shortening, along with posterior wall thickness. Using Western blot analysis, we confirmed that HIF-1α is significantly upregulated in the heart, whereas there were no changes in its regulatory proteins, prolyl hydroxylase domain-containing protein 2 (PHD2) and von Hippel-Lindau protein (VHL). PHD2 requires both oxygen and iron as cofactors for the hydroxylation of HIF-1α, marking it for ubiquination via VHL and subsequent destruction by the proteasome complex. We examined venous blood gas values in the tumor-bearing mice and found significantly lower oxygen concentration compared with control animals in the third week after tumor inoculation. We also examined select skeletal muscles to determine whether they are similarly affected. In the diaphragm, extensor digitorum longus, and soleus, we found significantly increased HIF-1α in tumor-bearing mice, indicating a hypoxic response, not only in the heart, but also in skeletal muscle. These results indicate that HIF-1α may contribute, in part, to the metabolic changes that occur during cancer cachexia.NEW & NOTEWORTHY We used proteomics and metadata analysis software to identify contributors to metabolic changes in striated muscle during cancer cachexia. We found increased expression of hypoxia-inducible factor-1α in the heart and skeletal muscle, suggesting a potential target for the therapeutic treatment of cancer cachexia.

Minocycline attenuates cardiac dysfunction in tumor-burdened mice.

Cardiovascular dysfunction as a result of tumor burden is becoming a recognized complication; however, the mechanisms remain unknown. A murine model of cancer cachexia has shown marked increases of matrix metalloproteinases (MMPs), known mediators of cardiac remodeling, in the left ventricle. The extent to which MMPs are involved in remodeling remains obscured. To this end a common antibiotic, minocycline, with MMP inhibitory properties was used to elucidate MMP involvement in tumor induced cardiovascular dysfunction. Tumor-bearing mice showed decreased cardiac function with reduced posterior wall thickness (PWTs) during systole, increased MMP and collagen expression consistent with fibrotic remodeling. Administration of minocycline preserved cardiac function in tumor bearing mice and decreased collagen RNA expression in the left ventricle. MMP protein levels were unaffected by minocycline administration, with the exception of MMP-9, indicating minocycline inhibition mechanisms are directly affecting MMP activity. Cancer induced cardiovascular dysfunction is an increasing concern; novel therapeutics are needed to prevent cardiac complications. Minocycline is a well-known antibiotic and recently has been shown to possess MMP inhibitory properties. Our findings presented here show that minocycline could represent a novel use for a long established drug in the prevention and treatment of cancer induced cardiovascular dysfunction.

Losartan treatment attenuates tumor-induced myocardial dysfunction.

UNLABELLED: Fatigue and muscle wasting are common symptoms experienced by cancer patients. Data from animal models demonstrate that angiotensin is involved in tumor-induced muscle wasting, and that tumor growth can independently affect myocardial function, which could contribute to fatigue in cancer patients. In clinical studies, inhibitors of angiotensin converting enzyme (ACE) can prevent the development of chemotherapy-induced cardiovascular dysfunction, suggesting a mechanistic role for the renin-angiotensin-aldosterone system (RAAS). In the present study, we investigated whether an angiotensin (AT) 1-receptor antagonist could prevent the development of tumor-associated myocardial dysfunction. METHODS AND RESULTS: Colon26 adenocarcinoma (c26) cells were implanted into female CD2F1 mice at 8weeks of age. Simultaneously, mice were administered Losartan (10mg/kg) daily via their drinking water. In vivo echocardiography, blood pressure, in vitro cardiomyocyte function, cell proliferation assays, and measures of systemic inflammation and myocardial protein degradation were performed 19days following tumor cell injection. Losartan treatment prevented tumor-induced loss of muscle mass and in vitro c26 cell proliferation, decreased tumor weight, and attenuated myocardial expression of interleukin-6. Furthermore, Losartan treatment mitigated tumor-associated alterations in calcium signaling in cardiomyocytes, which was associated with improved myocyte contraction velocity, systolic function, and blood pressures in the hearts of tumor-bearing mice. CONCLUSIONS: These data suggest that Losartan may mitigate tumor-induced myocardial dysfunction and inflammation.

Metalloproteinase expression is altered in cardiac and skeletal muscle in cancer cachexia.

Cardiac and skeletal muscle dysfunction is a recognized effect of cancer-induced cachexia, with alterations in heart function leading to heart failure and negatively impacting patient morbidity. Cachexia is a complex and multifaceted disease state with several potential contributors to cardiac and skeletal muscle dysfunction. Matrix metalloproteinases (MMPs) are a family of enzymes capable of degrading components of the extracellular matrix (ECM). Changes to the ECM cause disruption both in the connections between cells at the basement membrane and in cell-to-cell interactions. In the present study, we used a murine model of C26 adenocarcinoma-induced cancer cachexia to determine changes in MMP gene and protein expression in cardiac and skeletal muscle. We analyzed MMP-2, MMP-3, MMP-9, and MMP-14 as they have been shown to contribute to both cardiac and skeletal muscle ECM changes and, thereby, to pathology in models of heart failure and muscular dystrophy. In our model, cardiac and skeletal muscles showed a significant increase in RNA and protein levels of several MMPs and tissue inhibitors of metalloproteinases. Cardiac muscle showed significant protein increases in MMP-2, MMP-3, MMP-9, and MMP-14, whereas skeletal muscles showed increases in MMP-2, MMP-3, and MMP-14. Furthermore, collagen deposition was increased after C26 adenocarcinoma-induced cancer cachexia as indicated by an increased left ventricular picrosirius red-positive-stained area. Increases in serum hydroxyproline suggest increased collagen turnover, implicating skeletal muscle remodeling. Our findings demonstrate that cancer cachexia-associated matrix remodeling results in cardiac fibrosis and possible skeletal muscle remodeling. With these findings, MMPs represent a possible therapeutic target for the treatment of cancer-induced cachexia.

Mass Cytometry as a Tool for Investigating Senescence in Multiple Model Systems.

Cellular senescence is a durable cell cycle arrest as a result of the finite proliferative capacity of cells. Senescence responds to both intrinsic and extrinsic cellular stresses, such as aging, mitochondrial dysfunction, irradiation, and chemotherapy. Here, we report on the use of mass cytometry (MC) to analyze multiple model systems and demonstrate MC as a platform for senescence analysis at the single-cell level. We demonstrate changes to p16 expression, cell cycling fraction, and histone tail modifications in several established senescent model systems and using isolated human T cells. In bone marrow mesenchymal stromal cells (BMSCs), we show increased p16 expression with subsequent passage as well as a reduction in cycling cells and open chromatin marks. In WI-38 cells, we demonstrate increased p16 expression with both culture-induced senescence and oxidative stress-induced senescence (OSIS). We also use Wanderlust, a trajectory analysis tool, to demonstrate how p16 expression changes with histone tail modifications and cell cycle proteins. Finally, we demonstrate that repetitive stimulation of human T cells with CD3/CD28 beads induces an exhausted phenotype with increased p16 expression. This p16-expressing population exhibited higher expression of exhaustion markers such as EOMES and TOX. This work demonstrates that MC is a useful platform for studying senescence at a single-cell protein level, and is capable of measuring multiple markers of senescence at once with high confidence, thereby improving our understanding of senescent pathways.

Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV.

Human primary natural killer (NK) cells are being widely advanced for cancer immunotherapy. However, methods for gene editing of these cells have suffered low transduction rates, high cell death, and loss of transgene expression after expansion. Here, we developed a highly efficient method for site-specific gene insertion in NK cells using CRISPR (Cas9/RNP) and AAVs. We compared AAV vectors designed to mediate gene insertion by different DNA repair mechanisms, homology arm lengths, and virus concentrations. We then validated the method for site-directed gene insertion of CD33-specific CARs into primary human NK cells. CAR transduction was efficient, its expression remained stable after expansion, and it improved efficacy against AML targets.

Mass Cytometry, Imaging Mass Cytometry, and Multiplexed Ion Beam Imaging Use in a Clinical Setting.

Mass cytometry (MC), imaging mass cytometry (IMC), and multiplexed ion beam imaging (MIBI) represent a new generation of tools to understand increasingly complex systems. Although these technologies differ in their intended applications, with MC being most similar to flow cytometry, and IMC/MIBI being similar to immunohistochemistry, they all share a time of flight mass spectrometry (TOF MS) platform. These TOF MS platforms use metal conjugated antibodies as opposed to fluorophores, increasing the measurable parameters up to approximately 50 with a theoretic limit approximately 100 parameters. These tools are being adapted to understand highly complex systems in basic and clinical research.

Use of the Pyrimidine Analog, 5-Iodo-2'-Deoxyuridine (IdU) with Cell Cycle Markers to establish Cell Cycle Phases in a Mass Cytometry Platform.

The regulation of cell cycle phase is an important aspect of cellular proliferation and homeostasis. Disruption of the regulatory mechanisms governing the cell cycle is a feature of a number of diseases, including cancer. Study of the cell cycle necessitates the ability to define the number of cells in each portion of cell cycle progression as well as to clearly delineate between each cell cycle phase. The advent of mass cytometry (MCM) provides tremendous potential for high throughput single cell analysis through direct measurements of elemental isotopes, and the development of a method to measure the cell cycle state by MCM further extends the utility of MCM. Here we describe a method that directly measures 5-iodo-2'-deoxyuridine (IdU), similar to 5-bromo-2´-deoxyuridine (BrdU), in an MCM system. Use of this IdU-based MCM provides several advantages. First, IdU is rapidly incorporated into DNA during its synthesis, allowing reliable measurement of cells in the S-phase with incubations as short as 10-15 minutes. Second, IdU is measured without the need for secondary antibodies or the need for DNA degradation. Third, IdU staining can be easily combined with measurement of cyclin B1, phosphorylated retinoblastoma protein (pRb), and phosphorylated histone H3 (pHH3), which collectively provides clear delineation of the five cell cycle phases. Combination of these cell cycle markers with the high number of parameters possible with MCM allow combination with numerous other metrics.

PRMT5 regulates T cell interferon response and is a target for acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is a T cell-mediated immunological disorder and the leading cause of nonrelapse mortality in patients who receive allogeneic hematopoietic cell transplants. Based on recent observations that protein arginine methyltransferase 5 (PRMT5) and arginine methylation are upregulated in activated memory T cells, we hypothesized that PRMT5 is involved in the pathogenesis of aGVHD. Here, we show that PRMT5 expression and enzymatic activity were upregulated in activated T cells in vitro and in T cells from mice developing aGVHD after allogeneic transplant. PRMT5 expression was also upregulated in T cells of patients who developed aGVHD after allogeneic hematopoietic cell transplant compared with those who did not develop aGVHD. PRMT5 inhibition using a selective small-molecule inhibitor (C220) substantially reduced mouse and human allogeneic T cell proliferation and inflammatory IFN-γ and IL-17 cytokine production. Administration of PRMT5 small-molecule inhibitors substantially improves survival, reducing disease incidence and clinical severity in mouse models of aGVHD without adversely affecting engraftment. Importantly, we show that PRMT5 inhibition retained the beneficial graft-versus-leukemia effect by maintaining cytotoxic CD8+ T cell responses. Mechanistically, we show that PRMT5 inhibition potently reduced STAT1 phosphorylation as well as transcription of proinflammatory genes, including interferon-stimulated genes and IL-17. Additionally, PRMT5 inhibition deregulates the cell cycle in activated T cells and disrupts signaling by affecting ERK1/2 phosphorylation. Thus, we have identified PRMT5 as a regulator of T cell responses and as a therapeutic target in aGVHD.

Storage Conditions and Passages Alter IL-6 secretion in C26 adenocarcinoma cell lines.

The C26 adenocarcinoma tumor line is frequently used to establish peripheral tumors in mice for the study of cancer cachexia and cancer-related fatigue. Recently, we have noticed a progressive decline in the effects of tumor growth on our biological and behavioral measures in the tumor-bearing mice. Therefore, we compared effects of three aliquots of the C26 tumor cell line that differed in storage condition and number of passages on cytokine secretion, tumor growth, weight loss and fatigue behavior. Three aliquots of the C26 tumor line were selected as alpha (α), beta (ß), and gamma (γ). Aliquot α was an original C26 stock line that had been stored at -80°C. Aliquot ß was stored in liquid nitrogen. Aliquot γ was taken from aliquot ß and passaged three times. The three aliquots of the C26 tumor line showed differences in IL-6 mRNA and protein secretion in vitro, with aliquot ß showing the greatest IL-6 secretion. These differences were mirrored in vivo. Plasma IL-6 levels were elevated in all tumor bearing mice but was greatest in group ß mice. Carcass weight was decreased in all three tumor groups. Brain expression of IL-1ß mRNA was greatest in group ß and group ß demonstrated the greatest decline in running activity at day 19. Storage conditions and number of passages influence C26 tumor cell secretion of cytokines.Variations in C26 aliquots may explain differences observed between laboratories using the same cell line.We recommend always storing cell lines in liquid nitrogen and limiting the number of passages before use in experiments.

Ibuprofen ameliorates fatigue- and depressive-like behavior in tumor-bearing mice.

AIMS: Cancer-related fatigue (CRF) is often accompanied by depressed mood, both of which reduce functional status and quality of life. Research suggests that increased expression of pro-inflammatory cytokines is associated with skeletal muscle wasting and depressive- and fatigue-like behaviors in rodents and cancer patients. We have previously shown that treatment with ibuprofen, a nonsteroidal anti-inflammatory drug, preserved muscle mass in tumor-bearing mice. Therefore, the purpose of the present study was to determine the behavioral effects of ibuprofen in a mouse model of CRF. MAIN METHODS: Mice were injected with colon-26 adenocarcinoma cells and treated with ibuprofen (10mg/kg) in the drinking water. Depressive-like behavior was determined using the forced swim test (FST). Fatigue-like behaviors were determined using voluntary wheel running activity (VWRA) and grip strength. The hippocampus, gastrocnemius muscle, and serum were collected for cytokine analysis. KEY FINDINGS: Tumor-bearing mice showed depressive-like behavior in the FST, which was not observed in mice treated with ibuprofen. VWRA and grip strength declined in tumor-bearing mice, and ibuprofen attenuated this decline. Tumor-bearing mice had decreased gastrocnemius muscle mass and increased expression of IL-6, MAFBx and MuRF mRNA, biomarkers of protein degradation, in the muscle. Expression of IL-1ß and IL-6 was also increased in the hippocampus. Treatment with ibuprofen improved muscle mass and reduced cytokine expression in both the muscle and hippocampus of tumor-bearing mice. SIGNIFICANCE: Ibuprofen treatment reduced skeletal muscle wasting, inflammation in the brain, and fatigue- and depressive-like behavior in tumor-bearing mice. Therefore, ibuprofen warrants evaluation as an adjuvant treatment for CRF.

Fluoxetine prevents the development of depressive-like behavior in a mouse model of cancer related fatigue.

Cancer patients frequently suffer from fatigue, a complex syndrome associated with tiredness and depressed mood. Cancer-related fatigue (CRF) can be present at the time of diagnosis, escalates during treatment, and can persist for years after treatment. CRF negatively influences quality of life, limits functional independence, and is associated with decreased survival in patients with incurable disease. We have previously shown that increased pro-inflammatory cytokine expression in the brain contributes to depressive- and fatigue-like behaviors in a mouse model of CRF. Inflammatory cytokines increase the activity of indoleamine 2,3-dioxygenase (IDO) and kynurenine 3-monooxygenase (KMO), which competitively reduce serotonin synthesis. Reduced serotonin availability in the brain and increased production of alternative neuroactive metabolites of tryptophan are thought to contribute to the development of depression and fatigue. The purpose of this study was to determine the effects of fluoxetine, a selective serotonin reuptake inhibitor (SSRI), on brain cytokines and behavioral measures of fatigue and depression in tumor-bearing mice. Here we show that tumor growth increased brain expression of pro-inflammatory cytokines and KMO. Treatment with fluoxetine had no effect on tumor growth, muscle wasting, fatigue behavior, or cytokine expression in the brain. Fluoxetine, however, reduced depressive-like behaviors in tumor bearing mice. In conclusion, our data confirm that increased brain expression of pro-inflammatory cytokines is associated with tumor-induced fatigue- and depressive-like behaviors. However, it is possible to separate the effects of tumor growth on mood and fatigue-like behaviors using SSRIs such as fluoxetine.