Kunju Sridhar

Hematopoietic stem cell and progenitor cell mechanisms in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of disorders characterized by variable cytopenias and ineffective hematopoiesis. Hematopoietic stem cells (HSCs) and myeloid progenitors in MDS have not been extensively characterized. We transplanted purified human HSCs from MDS samples into immunodeficient mice and show that HSCs are the disease-initiating cells in MDS. We identify a recurrent loss of granulocyte-macrophage progenitors (GMPs) in the bone marrow of low risk MDS patients that can distinguish low risk MDS from clinical mimics, thus providing a simple diagnostic tool. The loss of GMPs is likely due to increased apoptosis and increased phagocytosis, the latter due to the up-regulation of cell surface calreticulin, a prophagocytic marker. Blocking calreticulin on low risk MDS myeloid progenitors rescues them from phagocytosis in vitro. However, in the high-risk refractory anemia with excess blasts (RAEB) stages of MDS, the GMP population is increased in frequency compared with normal, and myeloid progenitors evade phagocytosis due to up-regulation of CD47, an antiphagocytic marker. Blocking CD47 leads to the selective phagocytosis of this population. We propose that MDS HSCs compete with normal HSCs in the patients by increasing their frequency at the expense of normal hematopoiesis, that the loss of MDS myeloid progenitors by programmed cell death and programmed cell removal are, in part, responsible for the cytopenias, and that up-regulation of the “don’t eat me” signal CD47 on MDS myeloid progenitors is an important transition step leading from low risk MDS to high risk MDS and, possibly, to acute myeloid leukemia.

Protective effects of transient HO-1 overexpression on susceptibility to oxygen toxicity in lung cells

Rat fetal lung cells (RFL-6) were transiently transfected with a full-length rat heme oxygenase (HO)-1 cDNA construct and then exposed to hyperoxia (95% O2-5% CO2) for 48 h. Total HO activity and HO-1 protein were measured as well as cell viability, lactate dehydrogenase (LDH) release, protein oxidation, lipid peroxidation, and total glutathione to measure oxidative injury. HO-1 overexpression resulted in increased total HO activity (2-fold), increased HO-1 protein (1.5-fold), and increased cell proliferation. Immunohistochemistry revealed perinuclear HO-1 localization, followed by migration to the nucleus by day 3. Decreased cell death, protein oxidation, and lipid peroxidation but increased LDH release and glutathione depletion were seen with HO-1 overexpression. Reactive iron content could not explain the apparent loss of cell membrane integrity. With the addition of tin mesoporphyrin, total HO activity was decreased and all changes in injury parameters were normalized to control values. We conclude that moderate overexpression of HO-1 is protective against oxidative injury, but we speculate that there is a beneficial threshold of HO-1 expression.Rat fetal lung cells (RFL-6) were transiently transfected with a full-length rat heme oxygenase (HO)-1 cDNA construct and then exposed to hyperoxia (95% O2-5% CO2) for 48 h. Total HO activity and HO-1 protein were measured as well as cell viability, lactate dehydrogenase (LDH) release, protein oxidation, lipid peroxidation, and total glutathione to measure oxidative injury. HO-1 overexpression resulted in increased total HO activity (2-fold), increased HO-1 protein (1.5-fold), and increased cell proliferation. Immunohistochemistry revealed perinuclear HO-1 localization, followed by migration to the nucleus by day 3. Decreased cell death, protein oxidation, and lipid peroxidation but increased LDH release and glutathione depletion were seen with HO-1 overexpression. Reactive iron content could not explain the apparent loss of cell membrane integrity. With the addition of tin mesoporphyrin, total HO activity was decreased and all changes in injury parameters were normalized to control values. We conclude that moderate overexpression of HO-1 is protective against oxidative injury, but we speculate that there is a beneficial threshold of HO-1 expression.

Treatment of higher risk myelodysplastic syndrome patients unresponsive to hypomethylating agents with ON 01910.Na

In a Phase I/II clinical trial, 13 higher risk red blood cell-dependent myelodysplastic syndrome (MDS) patients unresponsive to hypomethylating therapy were treated with the multikinase inhibitor ON 01910.Na. Responses occurred in all morphologic, prognostic risk and cytogenetic subgroups, including four patients with marrow complete responses among eight with stable disease, associated with good drug tolerance. In a subset of patients, a novel nanoscale immunoassay showed substantially decreased AKT2 phosphorylation in CD34+ marrow cells from patients responding to therapy but not those who progressed on therapy. These data demonstrate encouraging efficacy and drug tolerance with ON 01910.Na treatment of higher risk MDS patients.

Specific Plasma Autoantibody Reactivity in Myelodysplastic Syndromes

Increased autoantibody reactivity in plasma from Myelodysplastic Syndromes (MDS) patients may provide novel disease signatures, and possible early detection. In a two-stage study we investigated Immunoglobulin G reactivity in plasma from MDS, Acute Myeloid Leukemia post MDS patients, and a healthy cohort. In exploratory Stage I we utilized high-throughput protein arrays to identify 35 high-interest proteins showing increased reactivity in patient subgroups compared to healthy controls. In validation Stage II we designed new arrays focusing on 25 of the proteins identified in Stage I and expanded the initial cohort. We validated increased antibody reactivity against AKT3, FCGR3A and ARL8B in patients, which enabled sample classification into stable MDS and healthy individuals. We also detected elevated AKT3 protein levels in MDS patient plasma. The discovery of increased specific autoantibody reactivity in MDS patients, provides molecular signatures for classification, supplementing existing risk categorizations, and may enhance diagnostic and prognostic capabilities for MDS.

Nanoscale approaches to define biologic signatures and measure proteomic response to targeted therapies in hematologic and solid tumors

The ability to quantify changes in protein activation in a clinical setting is important for the development of therapeutics that target cancer signaling pathways. A barrier to analyzing proteomic response in patients is the large amount of specimen required to make measurements at each time point. To overcome this barrier, we have developed the use of a highly sensitive microfluidic nano-immunoassay system (NIA) to quantify unphosphorylated, single- and multiple phosphorylated isoforms of proteins in patient specimens collected by minimally invasive blood draws or fine needle aspirates. We first developed tissue collection, handling and processing protocols to produce reliable NIA results using as little as 4 nanoliters of lysate. We next developed assays to measure over 40 proteomic parameters, including changes in proteins that mediate RAS and MAP Kinase signaling, cell cycle and apoptosis. To investigate the diagnostic potential of NIA, we have collected over 300 specimens from normal controls and patients with hematopoietic and solid tumors. We found that phosphoprotein profiles in tumor cells could distinguish tumor from normal cells. Further, patient tumors could be grouped based upon different patterns of percent ERK and MEK phosphorylation. In a clinical trial setting, we have now used NIA in conjunction with multicolor intracellular flow cytometry (FACS) to monitor changes in phospho-profiles in patients with receiving novel agents for hematopoietic cancers. In a prospective study of a novel cell cycle inhibitor in myelodysplastic syndrome (MDS), we have used NIA to measure changes in MAPK and cell cycle proteins in patient leukocytes sampled throughout serial time points during the study. In a prospective study of atorvastatin in patients with non-Hodgkin9s lymphoma, we have used NIA and FACS to distinguish three patterns of signaling changes in patient tumor cells, monocytes, and T cells. First, pathways that were initially activated in tumor cells decreased upon treatment with atorvastatin, with up to a 70% decrease in phospho-STAT3 and phospho-STAT5. Second, pathways that were initially suppressed in tumor cells (including phospho-PLC), normalized upon treatment. Third, expression levels of proteins in apoptotic pathways, including p38, increased upon atorvastatin treatment. Finally, changes in signaling associated with atorvastatin were found to be cell specific: effects on tumor cells were distinct from effects on non-tumor T cells and monocytes. Our studies demonstrate that NIA and FACS are complementary proteomic methods that can be used to quantify changes in cell signaling and distinguish responses in different cellular populations from very small clinical specimens sampled during therapeutic interventions. Nanoscale analysis of patient specimens is a feasible and promising approach for the development of new diagnostic markers and biomarkers of therapeutic activity. This talk is also presented as Poster A21.

Abstract B178: Nanoscale quantification of phosphorylated and unphosphorylated ERK and MEK isoforms differentiates tumor and nontumor clinical specimens

The MAP Kinase pathway is commonly dysregulated in cancer. We have developed the use of a highly sensitive microfluidic nano‐immunoassay system (NIA) to perform detailed analysis of ERK and MEK activation in hematopoietic and solid tumors. We recently reported use of NIA for measurement of proteins in as little as 4 nanoliters of lysate from preclinical and clinical lymphoma and leukemia specimens (Fan et al, Nature Medicine 2009). Now, we present results measuring specific isoforms of MAP Kinase proteins in cells sampled from patients with solid tumors and Myelodysplastic Syndrome (MDS). First, we developed tissue collection, handling and processing protocols to produce reliable NIA results in a small number of cells obtained by minimally invasive techniques. We collected over 100 blood buffy coat and fine needle aspirate (FNA) specimens from normal controls and patients with MDS or solid tumors. Using a single antibody that recognizes both the phosphorylated and unphosphorylated isoforms of ERK, we can determine not only relative levels of phosphorylated isoforms of ERK protein, but also calculate the percent phosphorylation of ERK in each clinical specimen. We have developed a similar method to quantify MEK isoforms. NIA revealed that adenocarcinoma, squamous cell carcinoma, renal cell carcinoma, and MDS samples could be distinguished from non‐tumor specimens based upon different patterns of phosphorylated ERK isoforms. To determine if signaling changes can be detected by NIA upon treatment of cells with novel targeted therapies, human leukemic TF1 cells were treated with a polo‐1 kinase (PLK‐1) inhibitor, ON01910 (Onconova, Inc.). We found the surprising result that only 5% of ERK1 and ERK2 were found to be phosphorylated in the TF1 cell line, whereas 45% and 90% of MEK1 and MEK2 were phosphorylated, respectively. Additionally, NIA analysis revealed a 20% decrease in the percent of phosphorylated MEK2 after treatment with ON01910. Taken together, our studies demonstrate that NIA can be used to identify specific changes in MAPK signaling pathway that distinguish normal from malignant cells as well as quantitatively measure the response of malignant cells to a targeted therapeutic. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B178.

EFFECT OF VARIABLE HEME OXYGENASE (HO-1) EXPRESSION ON OXYGEN RESISTANCE IN CULTURED HAMSTER FIBROBLASTS. ▴ 1212

EFFECT OF VARIABLE HEME OXYGENASE (HO-1) EXPRESSION ON OXYGEN RESISTANCE IN CULTURED HAMSTER FIBROBLASTS. ▴ 1212

Distinct transcriptomic and exomic abnormalities within myelodysplastic syndrome marrow cells

Abstract To provide biologic insights into mechanisms underlying myelodysplastic syndromes (MDS) we evaluated the CD34+ marrow cells transcriptome using high-throughput RNA sequencing (RNA-Seq). We demonstrated significant differential gene expression profiles (GEPs) between MDS and normal and identified 41 disease classifier genes. Additionally, two main clusters of GEPs distinguished patients based on their major clinical features, particularly between those whose disease remained stable versus patients who transformed into acute myeloid leukemia within 12 months. The genes whose expression was associated with disease outcome were involved in functional pathways and biologic processes highly relevant for MDS. Combined with exomic analysis we identified differential isoform usage of genes in MDS mutational subgroups, with consequent dysregulation of distinct biologic functions. This combination of clinical, transcriptomic and exomic findings provides valuable understanding of mechanisms underlying MDS and its progression to a more aggressive stage and also facilitates prognostic characterization of MDS patients.

MATURATIONAL DIFFERENCES IN EXPRESSION AND REGULATION OF LUNG HEME OXYGENASE IN HYPEROXIA. |[dagger]| 1052

Heme oxygenase (HO), the rate limiting enzyme in bilirubin production, is inducible by oxidant stresses including hyperoxia(O2)(1). In O2, newly born animals (<12 hrs. old:NEO) regulate HO differently than adults, who increase lung HO-1 mRNA in O2,(1), whereas NEO do not(2), despite increased HO activity. These differences may represent a maturational response. Therefore, Wistar rats (1-3 weeks:young) and adults (2 months) were exposed to 02 for 3 days. On Days 0 and 3 of exposure, lungs were evaluated for total HO activity (gas chromatography), HO-1 protein (Western), and HO-1 mRNA (Northern). Total lung glutathione (GSH) activity was used as an index of oxygen toxicity.Table (values expressed as mean% of Day 0 value±SE of 3-6 samples, *p=0.06 vs. air, †p<0.05 vs. air,Ψ p<0.01 vs. air). An 8.6 fold increase in HO-1 RNA was associated with a decrease in GSH in adults. No differences in HO-1 RNA were seen in young, but 1 week old rats showed a 2.2 fold increase in total HO activity, (p=0.06). This suggests a post-transcriptional regulation of HO-1, as previously shown with NEO. We conclude that there are maturational differences in the regulation of HO-1 mRNA with O2 in the lung. We speculate that GSH depletion is a necessary trigger for transcriptional regulation of lung HO-1 in O2.