THERAPEUTIC POTENTIAL OF TARGETING REACTIVE OXYGEN SPECIES (ROS) STRESS IN MYELODYSPLASTIC SYNDROME (MDS)

Myelodysplastic syndromes (MDS) are a diverse group of clonal hematologic disorders characterized by ineffective blood cell production (hematopoiesis), dysplastic (abnormal) cell morphology in one or more hematopoietic lineages, and progression to acute myeloid leukemia (AML). The response rate to current FDA approved therapies is low and not durable. Just about 50% of MDS patients respond to these drug therapies and a majority of responders relapse within 2-3 years. Hence there is a compelling need to investigate new therapy options. We investigated the anticancer potential and possible underlying molecular mechanisms of action of a plant-derived compound, Withaferin A (WFA) in MDS. We utilized the MDS-L cell line model to test the efficacy of WFA both in vitro and in vivo. WFA exhibited potent but selective cytotoxicity to MDS-L cells as seen by a dose-dependent decrease in cell viability of these cells when treated with WFA whereas WFA had no apparent significant effect on the viability of normal primary human bone marrow cells. In addition, WFA significantly reduced engraftment of MDS-L cells in a xenotransplantation model. Through the use of microarray gene expression analysis, we identified that reactive oxygen species (ROS)-activated JNK/AP-1 signaling is a major pathway mediating apoptosis of MDS-L cells by WFA. Increase in ROS plays a central role in the cytotoxicity of WFA in MDS-L cells. Consistent with the finding that increase in ROS plays a central role in mediating WFA cytotoxicity in MDS-L cells, WFA did not increase ROS levels in normal bone marrow cells. Taken together, these results suggest that pharmacologic manipulation of redox biology could be exploited to selectively target malignant cells while sparing normal cells in MDS

Novel Role of Prostate Apoptosis Response-4 Tumor Suppressor in B-Cell Chronic Lymphocytic Leukemia

Prostate apoptosis response-4 (Par-4), a proapoptotic tumor suppressor protein, is downregulated in many cancers including renal cell carcinoma, glioblastoma, endometrial, and breast cancer. Par-4 induces apoptosis selectively in various types of cancer cells but not normal cells. We found that chronic lymphocytic leukemia (CLL) cells from human patients and from Eµ-Tcl1 mice constitutively express Par-4 in greater amounts than normal B-1 or B-2 cells. Interestingly, knockdown of Par-4 in human CLL-derived Mec-1 cells results in a robust increase in p21/WAF1 expression and decreased growth due to delayed G1-to-S cell-cycle transition. Lack of Par-4 also increased the expression of p21 and delayed CLL growth in Eμ-Tcl1 mice. Par-4 expression in CLL cells required constitutively active B-cell receptor (BCR) signaling, as inhibition of BCR signaling with US Food and Drug Administration (FDA)–approved drugs caused a decrease in Par-4 messenger RNA and protein, and an increase in apoptosis. In particular, activities of Lyn, a Src family kinase, spleen tyrosine kinase, and Bruton tyrosine kinase are required for Par-4 expression in CLL cells, suggesting a novel regulation of Par-4 through BCR signaling. Together, these results suggest that Par-4 may play a novel progrowth rather than proapoptotic role in CLL and could be targeted to enhance the therapeutic effects of BCR-signaling inhibitors

Oxidative Stress-Induced JNK/AP-1 Signaling is a Major Pathway Involved in Selective Apoptosis of Myelodysplastic Syndrome Cells by Withaferin-A

Myelodysplastic syndromes (MDS) are a diverse group of malignant clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, dysplastic cell morphology in one or more hematopoietic lineages, and a risk of progression to acute myeloid leukemia (AML). Approximately 50% of MDS patients respond to current FDA-approved drug therapies but a majority of responders relapse within 2-3 years. There is therefore a compelling need to identify potential new therapies for MDS treatment. We utilized the MDS-L cell line to investigate the anticancer potential and mechanisms of action of a plant-derived compound, Withaferin A (WFA), in MDS. WFA was potently cytotoxic to MDS-L cells but had no significant effect on the viability of normal human primary bone marrow cells. WFA also significantly reduced engraftment of MDS-L cells in a xenotransplantation model. Through transcriptome analysis, we identified reactive oxygen species (ROS)-activated JNK/AP-1 signaling as a major pathway mediating apoptosis of MDS-L cells by WFA. We conclude that the molecular mechanism mediating selective cytotoxicity of WFA on MDS-L cells is strongly associated with induction of ROS. Therefore, pharmacologic manipulation of redox biology could be exploited as a selective therapeutic target in MDS

Radiation Induced Apoptosis of Murine Bone Marrow Cells is Independent of Early Growth Response 1 (EGR1)

An understanding of how each individual 5q chromosome critical deleted region (CDR) gene contributes to malignant transformation would foster the development of much needed targeted therapies for the treatment of therapy related myeloid neoplasms (t-MNs). Early Growth Response 1 (EGR1) is a key transcriptional regulator of myeloid differentiation located within the 5q chromosome CDR that has been shown to regulate HSC (hematopoietic stem cell) quiescence as well as the master regulator of apoptosis—p53. Since resistance to apoptosis is a hallmark of malignant transformation, we investigated the role of EGR1 in apoptosis of bone marrow cells; a cell population from which myeloid malignancies arise. We evaluated radiation induced apoptosis of Egr1+/+ and Egr1-/- bone marrow cells in vitro and in vivo. EGR1 is not required for radiation induced apoptosis of murine bone marrow cells. Neither p53 mRNA (messenger RNA) nor protein expression is regulated by EGR1 in these cells. Radiation induced apoptosis of bone marrow cells by double strand DNA breaks induced p53 activation. These results suggest EGR1 dependent signaling mechanisms do not contribute to aberrant apoptosis of malignant cells in myeloid malignancies

Increased p53 protein expression in irradiated <i>Egr1</i>^<i>+/+</i> and <i>Egr1</i>^-/- BM-MNCs is independent of mRNA expression.

<p><b>(A)</b> Cell lysates from irradiated Lin–ve enriched BM-MNCs (2 Gy or 6 Gy) at different time points (15’, 30’ and 45’) after radiation exposure were used for immunoblotting for p53. A549 and H358 cell lysates were used as positive and negative controls respectively. (<b>B)</b> Intensities of p53 bands were normalized to β-actin. Values are expressed as densitometric ratios. (<b>C)</b> BM-MNCs were lysed 45’ or 60’ after irradiation (2 Gy or 6 Gy). The RNA extracted from these lysed cells was used to determine p53 mRNA expression by qRT-PCR. (<b>D</b>) Cell lysates from phorbol 12-myristate 13-acetate (PMA) stimulated (30ng/ml) or irradiated Lin–ve encriched <i>Egr1</i>^<i>+/+</i> BM-MNCs (2 Gy or 6 Gy) at different time points (60’) and (45’, 60’) respectively. Lysate of normal splenic wildtype B cells stimulated with PMA (30ng/ml) for 60’ was used as a positive control for EGR1 expression [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169767#pone.0169767.ref041" target="_blank">41</a>]. RNA extracted from wildtype BM-MNCs in (C) was used to determine EGR1 mRNA expression by qRT-PCR.</p

Ionizing radiation induced apoptosis of primary <i>Egr1</i>^<i>+/+</i> and <i>Egr1</i>^-/- BM-MNCs equally in vitro.

<p><b>(A)</b> Representative analysis of irradiated lineage negative (Lin–ve) enriched BM-MNCs by flow cytometry. BM-MNCs isolated from WT and <i>Egr-1</i> KO mice were enriched for Lin–ve cells (as described in the methods). After 2 days of cytokine stimulation (mSCF-50ng/ml, mIL3-10ng/ml, hIL6-10ng/ml), Lin–ve enriched BM-MNCs were left untreated, or exposed to 2 Gy or 6 Gy irradiation (1 × 10⁶/ml). 24Hrs after irradiation, cells were stained with c-KIT-APC, Sca-1-PB and streptavidin APC CY7 antibodies, and annexin-V-PE CY7 to identify LSK and apoptotic cells respectively. A minimum of 500,000 cells were collected per sample on the BD LSR II flow cytometer and the data was analyzed using the FlowJo single cell analysis software for the percentage of apoptotic cells (by annexin) in the various cell populations (all cells, lin–ve cells and LSK cells). <b>(B)</b> Annexin +ve cells in the BM-MNC, and gated subpopulations from WT and <i>Egr-</i>1 KO mice are shown. <b>(C)</b> BM-MNC were stained for cleaved caspase-3. Left panel shows representative flow cytometry profile of irradiated WT or KO BM-MNCs (clear histograms) overlaid on untreated BM-MNCs (gray histograms). Summary of data from triplicate cultures is shown in the right panel. Results represent mean ± SE of triplicate cultures. *Indicates p<0.05 comparing untreated cells to cells exposed to radiation. Results from one of two experiments with similar outcomes are shown.</p

Radiation activates the DSB DNA response pathway in <i>Egr1</i>^<i>+/+</i> and <i>Egr1</i>^-/- BM-MNCs.

<p><b>(A)</b> Left: γ-H2AX protein expression by immunoblotting in irradiated BM-MNCs 30’, 45’ and 60’ after radiation exposure (2 Gy or 6 Gy). Right: Intensities of γ-H2AX bands were normalized to β-actin and are expressed as densitometric ratios. <b>(B)</b> Left: p-Chk2 protein expression by immunoblotting in irradiated BM-MNCs at different times after radiation exposure (2 Gy or 6 Gy). Right: p-Chk2 band intensities were normalized to those of GAPDH and are expressed as densitometric ratios. Results are representative of two experiments.</p

<i>Egr1</i>^<i>+/+</i> and <i>Egr1</i>^-/- mice have comparable recovery kinetics of blood cells after sub-lethal TBI.

<p>Mice were exposed to 6.5 Gy TBI. Blood cells were enumerated at 3, 9, 15, 22, and 28 days after TBI by the HEMAVet 950FS automatic veterinary hematology analyzer. Baseline (day 0) measurement was done before irradiation. The results are expressed as the mean of blood cells concentration ± SE (n = 8 mice/ group). Results from one of two similar experiments are shown.</p

Radiation induces DNA DSBs in <i>Egr1</i>^<i>+/+</i> and <i>Egr1</i>^-/- BM-MNCs.

<p>Immunofluorescence analysis of irradiated BM-MNCs for γ-H2AX foci was performed as described in the methods. Representative immunofluorescence images of γ-H2AX foci in BM-MNCs 4Hrs after exposure to 6 Gy irradiation.</p