Ganchimeg Ishdorj

Lysophosphatidic Acid Protects Cancer Cells from Histone Deacetylase (HDAC) Inhibitor-induced Apoptosis through Activation of HDAC

Histone deacetylases (HDACs) catalyze the removal of acetyl groups from histones and contribute to transcriptional repression. In addition, the HDAC inhibitors induce apoptosis in cancer cells through alterations in histone acetylation and activation of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) apoptotic pathway. Lysophosphatidic acid (LPA) is a growth factor that promotes survival of cancer cells through activation of G protein-coupled receptors. Here we show that HDAC inhibitors can induce apoptosis through activation of the TRAIL apoptotic pathway, and LPA prevented HDAC inhibitor-induced apoptosis and increased TRAIL receptor DR4 (death receptor 4) protein expression. This was associated with increased HDAC1 recruitment to the DR4 promoter following LPA treatment and a reduction in HDAC inhibitor-induced histone acetylation in the DR4 promoter. In addition, LPA induces HDAC enzyme activity in a dose- and time-dependent manner, and this is associated with HDAC1 activation and increased binding of HDAC1 to HDAC2. Reducing the expression of HDAC1 significantly lowered LPA-induced HDAC activity and increased histone acetylation. LPA induction of HDAC activity was blocked by the LPA receptor antagonist, Ki16425, or by inhibiting receptor activation with pertussis toxin. Reducing the expression of the LPA receptor LPA1 also blocked LPA-induced HDAC activation. In addition, LPA reduced histone acetyltransferase enzymatic activity. Finally, LPA attenuated the ability of the HDAC inhibitor to reduce HDAC activity. Thus, LPA enhances survival of cancer cells by increasing HDAC activity and reducing histone acetylation.

Regulation of autophagy in hematological malignancies: role of reactive oxygen species

Abstract Hematological malignancies are characterized by the accumulation of lymphoid and myeloid cell types due to selective proliferation and survival in blood, bone marrow and lymph nodes. Treatments of hematological malignancies are often effective but eventually relapse, and drug resistance occurs. A better understanding of the mechanism of action of both chemotherapeutic drugs and drug resistance is required. Autophagy has been shown to regulate both cell survival and cell death, leading to both cancer development and tumor suppression. In addition, many chemotherapeutic drugs induce autophagy, leading to either drug resistance or cell death. Autophagy is regulated by signaling pathways such as p53 and by the production of reactive oxygen species (ROS). This review focuses on the regulation of autophagy in human hematologic malignancy leading to either cell survival or death. In addition, the role that ROS play in regulating autophagy and its implication for hematological cancers is discussed.

A novel spliced variant of the TIN2 shelterin is present in chronic lymphocytic leukemia

The shelterin proteins play important roles in telomere maintenance and genome stability. These proteins have been found to be mutated in many cancers including CLL. Herein, we demonstrate here the presence of a novel spliced isoform of TIN2S in chronic lymphocytic leukemia (CLL), related to deletion of exon 2 in the TIN2 gene. The expressions of spliced TIN2S mRNA varied widely in CLL and there was an inverse relationship between the mRNA levels of full-length TIN2S and the spliced moiety. Small amounts of spliced TIN2S were also observed in normal B cells but not in T cells. Spliced TIN2S appeared dysfunctional, as immunoprecipitation studies showed the typical association of TRF2 and TIN2 in normal lymphocytes but not in CLL cells. Moreover, whereas TRF2 localized to the nucleus in normal lymphocytes, it was present in both nuclei and cytoplasm in CLL cells. The levels of spliced TIN2S increased with age and in 3 of 8 patients increased over time. The presence of the spliced variant failed to be related to telomere length in CLL suggesting other functions for this protein. Further studies are required to determine the etiology and biological significance of this unique spliced TIN2S variant.

Abstract 2850: Role of shelterin proteins for telomere shortening in chronic lymphocytic leukemia

Telomeres are important in maintaining the integrity of the chromosomes. A series of shelterin proteins (TRF1, TRF2, RAP1, POT1a and b, TIPP1 and TIN2), are involved in the maintenance of telomeres. In chronic lymphocytic leukemia (CLL), the telomeres are shorter than in normal B cells, with very short telomeres being associated with poor survival. In the present study, we demonstrate that the leukemia cell telomere length shortens over time were CLL are followed over year, indicating that telomere shortening in these cells is an ongoing process. When CLL cell metaphases were examined by Q-FISH, telomere length shortening was found to affect all chromosomes equally. To determine whether these telomere changes were related to alterations in the shelterin complex, protein levels of the six shelterins were determined in CLL cells and normal pooled B cells. In general, apart from TIN2, the shelterin protein levels were increased in CLL cells and TRF2 levels were higher in IgVH unmutated cells than in mutated cells. TIN2 protein levels were dramatically reduced in CLL cells as compared to normal B cells and an alternative isoform of TIN2 was observed, both in IgVH mutated and unmuted cases. TIN2 targeted RT-PCR followed by sequencing revealed a deletion of the entire exon 2, totaling 105 bp. This abnormality appeared in monoclonal B-cell lymphocytosis (MBL) indicating that this was an early event. In addition, a subgroup of patients showed an increase in the spliced form of TIN2, when followed over time, suggesting that this might provide the cell with a survival advantage. A protein translated from the spliced variant RNA is expected to be deleted for 35 amino acids at the N-terminus, which is the binding site for TRF2. As TIN2 maintains telomere structure by forming a complex of TRF1 and TRF2 with the telomere, this should not occur with the splice variant. Indicating a pathological role of spliced TIN2 in CLL, TRF2 was localized in both the nuclei and cytoplasm in CLL cells compared to localization only to the nucleus in normal B cells. To confirm the function of the spliced TIN2, we have overexpressed the both full length and spliced clones of TIN2 in HEK293 cells and are presently determining the localization of TRF2 and the ability to form a TRF2/TIN2 complex. Thus, these studies demonstrate a unique splice variant of TIN2 in CLL, which potentially may influence the formation of the shelterin complex and contribute to telomere shortening in this disease. Citation Format: Ganchimeg Ishdorj, Sara EF Kost, Yunli Zang, Spencer B. Gibson, James B. Johnston. Role of shelterin proteins for telomere shortening in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2850.

Abstract 597: The combination of valproic acid and fludarabine treatment induces a synergistic apoptotic response in chronic lymphocytic leukemia (CLL) patients involving the lysosomal protease cathepsin B.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Chronic Lymphocytic Leukaemia (CLL) is the most common hematological malignancy in the western world. Fludarabine, a nucleoside analogue, is commonly used in combinational treatments for CLL. However, patients commonly develop resistance to fludarabine based therapies. Valproic Acid (VPA), an inhibitor of histone deacetylases (HDACs), showed synergistic apoptotic responses when combined with fludarabine in human leukemic cells and primary CLL cells. The mechanism for this synergistic apoptotic response is unknown. We found that fludarabine and VPA treatment increased activates caspases-8, -9, -3, and caspase-2 and a decreased in expression of anti-apoptotic proteins Mcl-1 and XIAP. Treatment with fludarabine alone or in combination with VPA led to the loss of lysosome integrity suggesting a leakage of the lysosomal content into the cytosol in response to these drugs. Chemical inhibition of a specific lysosomal protease, cathepsin B, using CA074-Me was sufficient to stabilize Mcl-1 and XIAP while reducing caspase activation and apoptosis. Addition of purified cathepsin B to leukemic cell lysates led to the reduction in protein levels of Mcl-1, XIAP and pro-caspase-2, thus suggesting that the re-localization of cathepsin B into the cytosol is sufficient to degrade these proteins. VPA treatment increased cathepsin B levels in both leukemic cell lines and primary CLL cells. VPA also increased cathepsin B activity. In addition, six relapsed CLL patients who had received at least one prior therapy with fludarabine were treated with VPA alone or in combination with fludarabine. No responses were seen after 28 days using VPA alone but in five patients who continued on VPA with fludarabine, three patients showed reduced lymphocyte count after receiving at most five cycles of the combination therapy. When the CLL cells from VPA-treated CLL patients were examined, VPA administration induced increased levels of histone-3 acetylation and cathepsin B expression in vivo. Thus, VPA and fludarabine synergistic apoptotic response is mediated by cathepsin B activation leading to a decrease in anti-apoptotic proteins in CLL cells and likely contributes to the clinical response observed in relapsed, fludarabine-refractory CLL patients. Citation Format: Ju Yoon Yoon, David Szwajcer, Ganchimeg Ishdorj, Pat Benjaminson, Rajat Kumar, James B. Johnston, Spencer B. Gibson. The combination of valproic acid and fludarabine treatment induces a synergistic apoptotic response in chronic lymphocytic leukemia (CLL) patients involving the lysosomal protease cathepsin B. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 597. doi:10.1158/1538-7445.AM2013-597

Abstract 4983: Correlation telomere shortening in chronic lymphocytic leukemia cells with oxidative stress and dysregulation of shelterin proteins

Telomeres are important in maintaining the integrity of the chromosomes. A series of shelterin proteins (TRF1, TRF2, RAP1, POT1a and b, TIPP1 and TIN2), are involved in the maintenance and synthesis of telomeres. In chronic lymphocytic leukemia (CLL), the telomeres are shorter than in normal B cells, with very short telomeres being associated with poor survival. The mechanism for this telomere shortening has been evaluated in the present study. We demonstrated in 150 CLL patients using a Q-RT-PCR assay that the median telomere length was similar in mutated IgV H (Mu-IgV H ) cases as in normal B cells, but was significantly shorter in unmutated (Un-IgV H ) cases. However, there was considerable variation and overlap in telomere length between the groups. In addition, telomere length was found to shorten in both IgVH mutated and unmutated groups when patients were sequentially followed over years indicating that telomere shortening was an ongoing process. When lymphocytes were stimulated and the telomeres evaluated in metaphase by Q-FISH, telomere length shortening affected all chromosomes equally. To determine if telomere shortening in CLL cells was related to increased free radical formation, we measured plasma lipid peroxidation level (Lipid Hydroperoxide (LPO) assay, Cayman) and DNA breaks (gamma H2AX). As compared to normal B cells, there was increased lipid peroxidation and DNA breaks in both mutated and unmutated CLL cells. When cells were treated with N-acetyl-L-cysteine to scavenge free radicals, there was a decrease in DNA breakage. To assess, whether these changes were associated with alterations of the shelterin complex, protein levels of the shelterins were measured. TRF1 and TRF2 protein levels were increased in all patients compared to normal B cells. TRF2 was also increased in the cytoplasm of CLL cells. Following drug induced apoptosis in CLL cells TRF2 levels decreased but not TRF1 levels. In summary, these studies suggest that the short telomeres in CLL may be related to increased oxidative stress in these cells. TRF1 and TRF2 levels were increased in CLL cells regardless of telomere length suggesting that abnormalities in these proteins may also be involved in telomere shortening. Ongoing studies are evaluating the mechanisms responsible for the shelterin changes in CLL cells and whether altering the levels of these proteins can influence cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4983. doi:1538-7445.AM2012-4983

Abstract 2651: Cucurbitacin I (JSI-124) activates the JNK signaling pathway leading to transcriptional up-regulation of vascular endothelial growth factor in B leukemia cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL JSI-124 (Cucurbitain I) is a biochemical compound isolated from the herbal plant families, which have been used as folk medicines for centuries in Asian countries. The molecular mechanisms for this inhibitor acts primarily through STAT3 inhibition in many tumors, including chronic lymphocytic leukemia. However, other molecular targets of JSI-124 are not fully understood. The objective of this study was to determine the molecular targets of JSI-124 beyond STAT3. Chemotherapeutic drugs often induce a stress response in cancer cells. One of early stress response pathways is the c-Jun N-terminal kinase (JNK) pathway. Herein, we show that JSI-124 activates JNK and increased the expression of c-Jun protein in the B leukemia cell lines BJAB, I-83 and NALM-6. Interestingly, the inhibition of the JNK signaling pathway failed to effect JSI-124 induced cell cycle arrest or apoptosis in these leukemia cells but repressed c-Jun level. Furthermore, JNK activation also failed to effect JSI-124 inhibition of STAT3 phosphorylation in these cells. It is known that one of the important factors involved in JNK signaling is Vascular Endothelial Growth Factor (VEGF). We found that JSI-124 treated BJAB cells showed an increased in mRNA and protein level in VEGF in BJAB, I-83 and NALM-6 cells. Knockdown of c-Jun expression and inhibition of JNK activation significantly blocked JSI-124 induced VEGF expression. Taken together, our data demonstrate that JSI-124 activates JNK signaling pathway leading to VEGF expression independent of inhibition of STAT3 activation. This could lead to increased angiogenesis in B leukemia cells treated with JSI-124. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2651. doi:10.1158/1538-7445.AM2011-2651