Metabolic Shift In Hypoxia-Adapted CML Cells and Mechanisms Of Acquired Dasatinib Resistance Uncovered By Proteomic iTRAQ Profiling

Abstract Hypoxia is one of the essential components of the leukemia bone marrow (BM) microenvironment that promotes leukemia cell homing, survival and chemoresistance (Benito, PlosOne 2011). Tyrosine kinase inhibitors (TKIs) do not eradicate the total mass of chronic myeloid leukemia (CML) cells including primitive and quiescent cells. Persistence of CML cells in the hypoxic BM niche after cessation of TKI therapy may result in disease relapse. We have reported that hypoxia adapted CML cells acquire TKI resistance associated with higher glyoxalase-1 (Glo-1) enzyme activity which detoxifies methylglyoxal, a cytotoxic by-product of glycolysis (Takeuchi, Cell Death Differ 2010). In this study, we employed a proteomic approach based on isobaric tags for relative and absolute quantification (iTRAQ, Applied Biosystems) to investigate additional molecular mechanisms of CML adaptation to hypoxia and acquired resistance to dasatinib under hypoxic conditions. The specific pathway alterations were identified by KEGG(Kyoto University) and MetaCore (GeneGo). We utilized two hypoxia-adapted (HA) subclones of CML cell lines, KCL22-HA and KBM5-HA cells, which were selected over a month under 1.0 % oxygen culture conditions. The growth rate of both HA-CML cell lines was slower than that of the corresponding parental cells (ratio of incremental increase in cell numbers, 0.39 for KCL22-HA/KCL22, 0.54 for KBM5-HA/KBM5 at 48hrs). Although parental KCL22 cells were sensitive to dasatinib, KCL22-HA cultured under hypoxia acquired resistance to dasatinib (IC50: KCL22 0.1 nM, KCL22-HA >20nM, at 48hrs by MTT). In contrast, dasatinib induced more prominent cell growth inhibition in KBM5-HA cells cultured under hypoxia compared to KBM5 parental cells (IC50: KBM5 1.3 nM, KBM-5/HA 0.3 nM). Dasatinib effectively downregulated the phosphorylation levels of Stat5 and ERK in parental KCL22 and KBM5 cells. Notably, the baseline levels of p-Stat5 and p-ERK were markedly diminished in both KCL22-HA and KBM5-HA cells. We next performed iTRAQ proteomic analysis and detected more than 1,300 proteins in each cell type. Comparison of the basal proteome of KCL22 vs KCL22-HA and KBM5 vs KBM5-HA cells showed differential expression of 54 proteins in KCL22 isogenic cells (37 upregulated, 17 downregulated) and of 159 proteins in KBM5 cells (56 upregulated, 103 downregulated). These alterations included consistent activation of glycolysis and gluconeogenesis pathway (p<0.0001) along with increased ATP synthase (p=0.02) and hypoxia induced HIF1 activation (p<0.0001) in both cells adapted to grow under hypoxia. In dasatinib-resistant KCL-HA cells, these changes were partially reversed by dasatinib which affected expression of 19 proteins (5 up-regulated / 14 down-regulated) with significant decrease in oxidative phosphorylation (p=0.01), ATP synthase (p=0.02), spliceosome (p=0.02) and Cytochrome C (p=0.04). On the contrary, in dasatinib-sensitive KBM5-HA cells dasatinib upregulated 296 proteins including 39 apoptogenic proteins such as Cytochrome C (p<0.001) and apoptosis related mitochondrial chaperones HSP10 and HSP60 (p<0.001), along with further upregulation of the enzymes involved in the glycolytic energy production (p<0.001) and stimulation of nuclear mRNA splicing via spliceosome (p<0.001), known to play a crucial role in the control of gene expression of the apoptosis-regulating genes. We observed the consistent upregulation of glycolysis and increases of ribosome and spliceosome after dasatinib treatment in dasatinib-sensitive parental KCL22 and KBM5 cells. Interestingly, dasatinib-resistant KBM5-STI cells harboring T315 Bcr-Abl mutation exhibited significantly lower levels of baseline gluconeogenesis (p<0.0001) and translation regulation of translation initiation pathways (p<0.0001) compared to the parental KBM5 cells. In summary, these findings demonstrate the dramatic metabolic changes in hypoxia adjusted CML cells and indicate that hypoxia adapted / dasatinib resistant CML cells repress their stimulated glycolic metabolic state and oxidative phosphorylation along with spliceosomal mRNA splicing after dasatinib exposure, which allow them to enter quiescent stage and escape from apoptosis. Altogether these data indicate novel mechanisms of acquired bone marrow microenvironment-mediated resistance to TKI in CML. Disclosures: Tabe: Bristol-Myers Squibb: Research Funding.

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P6277The impact of exercise training in combination with statin use on skeletal muscle mitochondrial oxidative phosphorylation and metabolomics in obese rats

European Heart Journal ◽

10.1093/eurheartj/ehz746.0876 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

I Urbaneck ◽

F Lorenz ◽

I Materzok ◽

L Maletzki ◽

M Pietzner ◽

...

Keyword(s):

Skeletal Muscle ◽

Cytochrome C ◽

Exercise Training ◽

Oxidative Phosphorylation ◽

Atp Synthase ◽

Statin Treatment ◽

Mitochondrial Oxidative Phosphorylation ◽

Complex Iv ◽

Obese Rats ◽

Statin Use

Abstract Background Exercise training (ET) and statin treatment both alter skeletal muscle function. Purpose We investigated the effects of a combined exercise and statin use on skeletal muscle mitochondrial oxidative phosphorylation (OxPhos) and metabolic alterations in obese rats. Methods Eight-week-old male Wistar rats were used. A total of 14 animals received standard chow, while 46 rats were fed a high-fat diet (HFD) for 20 weeks. After 8 weeks, the rats were randomized into 6 groups: sedentary (n=8), ET (n=6), sedentary with HFD (n=11), ET with HFD (n=11), statin with HFD (n=13) and ET with HFD and statins (n=11). Simvastatin (10mg/d/kg) was added to the drinking water. ET was performed for 12 weeks, 5 days/week for 1 h/day at 18 m/min in a motorized running wheel. OxPhos was assessed by complex-specific antibodies and targeted metabolomics using the Biocrates p180 kit. All experiments were done on frozen samples of the M. gastrocnemicus. An ANOVA with fixed effects for diet, exercise, statin treatment and statin-exercise interaction was used to identify significantly different metabolites. Results Statin use was associated with significantly lower cholesterol levels, but did not affect exercise duration and intensity compared to none-use. In sedentary animals, HFD increased OxPhos complex II (succinate dehydrogenase), complex IV (cytochrome-c-oxidase) and V (ATP synthase) while statin treatment diminished this increase in all complexes. HFD increased complex IV independent of statin treatment but had no effect on complex II and V in ET rats. Complex IV was increased due to ET only in HFD fed rats compared to rats on normal chow but decreased in contrast to sedentary animals on a HFD. With regards to metabolomics, we found 57 metabolites which were influenced by HFD while no metabolites were identified with a significant effect for ET. A significant statin-exercise interaction was found for three lysophosphatidylcholines (lysoPC a C26.0, lysoPC a C26.1, lysoPC a C24.0), one phosphatidylcholine (PC aa C42.6) and one sphingomyelin (SM C16.1). HFD decreased the concentration of all mentioned metabolites compared to standard chow fed animals. Likewise, ET increased the concentration of metabolites compared to sedentary animals on HFD. Statin treatment led to an increase, while statin in combination with ET did not rescue this effect. Conclusion HFD induced severely impaired skeletal muscle OxPhos independent of ET and statin treatment. Our findings suggest a limiting rate of NADH production in the tricarboxylic acid cycle as a potential mechanism. However, ET prevented the increase in cytochrome-c-oxidation while statins blocked the HFD induced increase in ATP synthase. Our metabolomics results imply that future research should consider the lipotoxic effects of a HFD when assessing skeletal muscle alterations due to ET or statins. Of particular interest could be the 5 metabolites that have been shown to be impacted by a statin-exercise interaction.

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Mesenchymal Stem Cells Self-Regulate Their Differentiation To Maintain the Bone Marrow Stromal System.

Blood ◽

10.1182/blood.v104.11.2335.2335 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2335-2335

Author(s):

Iekuni Oh ◽

Akira Miyazato ◽

Hiroyuki Mano ◽

Tadashi Nagai ◽

Kazuo Muroi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Cell Lines ◽

Molecular Mechanisms ◽

Adipocyte Differentiation ◽

Expression Profiles ◽

Self Renewal ◽

Hematopoietic Stem ◽

Steady Level

Abstract Mesenchymal stem cells (MSCs) account for a very small population in bone marrow stroma as a non-hematopoietic component with multipotency of differentiation into adipocytes, osteocytes and chondrocytes. MSC-derived cells are known to have hematopoiesis-supporting and immunomodulatory abilities. Although clinical applications of MSCs have already been conducted for the suppression of graft versus host disease in allogeneic stem cell transplantation and for tissue regeneration, underlying mechanisms of the biological events are still obscure. Previously, we established a differentiation model of MSCs using a mouse embryo fibroblast cell line, C3H10T1/2 (10T1/2) (Nishikawa M et al: Blood81:1184–1192, 1993). Preadipocyte (A54) and myoblast (M1601) cell lines were cloned by treatment with 5-azacytidine. A54 cells and M1601 cells can terminally differentiate into adipocytes and myotubes, respectively, under appropriate conditions, while parent 10T1/2 cells remain undifferentiated. Moreover, A54 cells show a higher ability to support hematopoiesis compared with the other cell lines. In this study, we analyzed gene expression profiles of the three cell lines by using DNA microarray and real-time PCR to investigate molecular mechanisms for maintaining immaturity of parent 10T1/2 cells. In A54 cells, 202 genes were up-regulated, including those encoding critical factors for hematopoiesis such as SCF, Angiopoietin-1, and SDF-1 as well as genes known to be involved in adipocyte differentiation such as C/EBPα, C/EBPδ and PPAR-γ genes. These data are consistent with the hematopoiesis-supporting ability of A54 cells. During adipocyte differentiation, SCF and SDF-1 expression levels decreased in A54 cells while C/EBPα expression showed a steady level. Recently, osteoblasts have been reported to play crucial roles in “niche” for self-renewal of hematopoietic stem cells. Our results also implicate that precursor cells of non-hematopoietic components may have important roles for hematopoiesis in bone marrow. Meanwhile, in parent 10T1/2 cells, 105 genes were up-regulated, including CD90, Dlk, Wnt5α and many functionally unknown genes. Although C/EBPα expression was induced in 10T1/2 cells without differentiation under the adipocyte differentiation conditions, CD90 expression decreased, Dlk showed a steady level and Wnt5α was up-regulated. Assuming that some regulatory mechanisms are needed to keep an immature state of parent 10T1/2 cells even under the differentiation-inducible conditions, we performed following experiments. First, enforced Dlk expression in A54 cells did not inhibit terminal differentiation to adipocytes under the differentiation conditions. Second, when we cultured A54 cells in the conditioned media of parent 10T1/2 cells under the differentiation-inducible conditions, adipocyte differentiation was inhibited, suggesting that 10T1/2 cells produce some soluble molecules that can inhibit adipocyte differentiation. Since Wnt family is known to be involved in the regulation of self-renewal of several stem cells, Wnt5α may be one candidate for maintenance of “stemness” of MSCs. Taken together, the data of 10T1/2 cells suggest that MSCs can self-regulate their differentiation in the bone marrow stromal system. This concept may be important to investigate the fatty change of bone marrow in aging and in aplastic anemia.

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Bone Marrow Stromal Cells Induce Bortezomib Resistance in Multiple Myeloma Cells through Downregulation of miRNA-101-3p Targeting Survivin

Blood ◽

10.1182/blood.v126.23.1772.1772 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1772-1772 ◽

Cited By ~ 3

Author(s):

Jahangir Abdi ◽

Yijun Yang ◽

Patrick Meyer-Erlach ◽

Hong Chang

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Drug Resistance ◽

Cell Lines ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Molecular Mechanisms ◽

Marrow Stromal Cells ◽

Survivin Gene ◽

Induced Apoptosis

Abstract INTRODUCTION It is not yet fully understood how bone marrow microenvironment components especially bone marrow stromal cells (BMSCs) induce drug resistance in multiple myeloma (MM). This form of drug resistance has been suggested to pave the way for intrinsic (de novo) resistance to therapy in early stages of the disease and contribute to acquired drug resistance in the course of treatment. Hence, deciphering the molecular mechanisms involved in induction of above resistance will help identify potential therapeutic targets in MM combined treatments. Our previous work showed that BMSCs (normal and MM patient-derived) induced resistance to bortezomib (BTZ) compared with MM cells in the absence of stroma. This resistance was associated with modulation of a transcriptome in MM cells, including prominent upregulation of oncogenes c-FOS, BIRC5 (survivin) and CCND1. However; whether these oncogenes mediate BTZ resistance in the context of BMSCs through interaction with miRNAs is not known. METHODS Human myeloma cell lines, 8226, U266 and MM.1s, were co-cultured with MM patient-derived BMSCs or an immortalized normal human line (HS-5) in the presence of 5nM BTZ for 24 h. MM cell monocultures treated with 5nM BTZ were used as controls. Co-cultures were then applied to magnetic cell separation (EasySep, Stem Cell Technologies) to isolate MM cells for downstream analyses (western blotting and qPCR). Total RNA including miRNAs was isolated from MM cell pellets (QIAGEN miRNeasy kit), cDNAs were synthesized (QIAGEN miScript RT II kit) and applied to miScript miRNA PCR Array (SABioscience, MIHS-114ZA). After normalization of all extracted Ct values to 5 different housekeeping genes, fold changes in miRNA expression were analyzed in co-cultures compared to MM cell monocultures using the 2-ΔΔCt algorithm. Moreover, survivin gene was silenced in MM cells using Ambion® Silencer® Select siRNA and Lipofectamine RNAiMAX transfection reagent. Survivin-silenced cells were then seeded on BMSCs and exposed to BTZ. Percent apoptosis of gated CD138+ MM cells was determined using FACS. For our overexpression and 3'UTR reporter experiments, we transiently transfected MM cells with pre-miR-101-3p, scrambled miRNA or pEZX-3'UTR constructs using Endofectin reagent (all from GeneCopoeia). RESULTS BMSCs upregulated survivin gene / protein (a member of inhibitors of apoptosis family) and modulated an array of miRNAs in MM cells compared to MM cells in the absence of stroma. The more noticeably downregulated miRNAs were hsa-miR-101-3p, hsa-miR-29b-3p, hsa-miR-32-5p, hsa-miR-16-5p (4-30 fold) and highly upregulated ones included hsa-miR-221-3p, hsa-miR-409-3p, hsa-miR-193a-5p, hsa-miR-125a-5p (80-330 fold). We focused on miRNA-101-3p as it showed the highest level of downregulation (30 fold) and has been shown to function as an important tumor suppressor in other malignancies. Real time RT-PCR confirmed downregulation of miRNA-101-3p. Moreover, microRNA Data Integration Portal (mirDIP) identified miRNA-101-3p as a putative target for survivin and Luciferase activity assays confirmed binding of miRNA-101-3p to 3'UTR of survivin. In addition, overexpression of miRNA-101-3p downregulated survivin and sensitized MM cells to BTZ-induced apoptosis. Furthermore, silencing of survivin upregulated miRNA-101-3p and increased BTZ-induced apoptosis in MM cell lines both in the absence of BMSCs (Apoptosis range in BTZ-treated conditions: 57.65% ± 4.91 and 28.66% ± 0.78 for si-survivin and scrambled control, respectively, p<0.05) and in the presence of BMSCs (41.23% ± 1.43 and 14.8% ± 0.66, for si-survivin and scrambled control, respectively, p<0.05). CONCLUSION Our results indicate that BMSCs downregulated miRNA-101-3p and upregulated survivin in MM cells compared to MM cells in the absence of stroma. Silencing of survivin or overexpression of miRNA-101-3p sensitized MM cells to BTZ in the presence of BMSCs. These findings suggest that miRNA-101-3p mediates BTZ response of MM cells in the presence of BMSCs by targeting survivin and disclose a role of survivin-miRNA-101-3p axis in regulation of BMSCs-induced BTZ resistance in MM cells, thus provide a rationale to further investigate the anti-myeloma activity of miRNA-101-3p in combination with BTZ as a potential novel therapeutic strategy in MM. Disclosures No relevant conflicts of interest to declare.

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A Gene-Targeted Mouse Model for Bone Marrow Failure Syndrome.

Blood ◽

10.1182/blood.v106.11.4235.4235 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4235-4235

Author(s):

Masataka Kasai ◽

Yuko Fukuda ◽

Reiko Ishida

Keyword(s):

Bone Marrow ◽

Transcription Factors ◽

Molecular Mechanisms ◽

Bone Marrow Failure ◽

Myeloid Cells ◽

Pcr Analysis ◽

Mrna Levels ◽

Control Of Gene Expression ◽

Wild Mice ◽

Immature Myeloid Cells

Abstract Bone marrow failure is a disease syndrome characterized by dysfunction of bone marrow to produce mature blood cells. However, molecular mechanisms causing the disease syndromes remain remarkably obscure. We generated mice homozygous for an inactivating mutation of the Translin gene. Most of the aged mutant mice (Translin−/−) were found to exhibit progressive bone marrow dysfunction manifested by a reduction in immature myeloid cells and erythroblasts, and eventually developed marked splenomegaly, with up to 30-fold elevation in weight. Histological examination of enlarged spleens revealed extramedullary hematopoiesis, prominent expansion of the red pulp areas with an increase in the number of mature granulocytes and a marked lack of immature myeloid cells. Furthermore, these mice also featured complete loss of immature myeloid cells and erythroblasts in bone marrow. On the other hand, an increase in the number of reticulocytes (over 10 %) and unusual appearance of metamyelocytes and orthochromatic erythroblasts were seen in peripheral blood. A cascade of the lineage-restricted transcription factors is known to determine developmental decisions regarding hematopoiesis. How levels of transcription factors are translated into a decision to control lineage commitment is an intriguing question in hematopoiesis which remains to be explored. Therefore, we considered the bone marrow failure in aged Translin−/− might be due to decreased expression of the Ets family transcription factor PU.1 that has previously been suggested to be essential for myeloid development. However, quantitative RT-PCR analysis showed PU.1 mRNA to be expressed at equivalent levels in bone marrows of mutant and wild mice. In contrast, our data indicated a sharp reduction of the mRNA levels of the basic helix-loop-helix (bHLH) protein, E2A and its dimerization partner, TAL1, suggesting a contribution of E47/TAL heterodimer having distinctive DNA-binding properties for fine tune control of gene expression during hematopoiesis. In conclusion, the present studies demonstrated that expression levels of Translin are crucial to the bone marrow’s functional ability, and that our findings will shed light on the molecular events involved in bone marrow failure syndromes.

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TNF-α Confers Resistance of Myeloma Cells to IMiDs through TRAF2 Degradation

Blood ◽

10.1182/blood-2019-123141 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1809-1809

Author(s):

Jiye Liu ◽

Teru Hideshima ◽

Lijie Xing ◽

Kenneth Wen ◽

Yu-Tzu Tai ◽

...

Keyword(s):

Patient Outcome ◽

Bone Marrow ◽

Proteasome Inhibitor ◽

Molecular Mechanisms ◽

Lentiviral Vector ◽

Acquired Resistance ◽

Proteasomal Degradation ◽

Interacting Protein ◽

Protein Levels ◽

Tnf Α

The development of novel agents including immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) has led to improved patient outcome in multiple myeloma (MM); however, acquired resistance to IMiDs commonly underlies relapse during the course of treatment. Previous studies show that IMiDs bind to the CRL4CRBN ubiquitin ligase cereblon (CRBN) and promote proteasomal degradation of IKZF1 and IKZF3 followed by downregulation of c-Myc and IRF4, resulting in MM cell growth inhibition. Therefore, CRBN is the primary binding target and master regulator of IMiDs sensitivity; however, the molecular mechanisms regulating resistance to IMiDs have not been fully defined. Importantly, some MM cells show resistance to IMiDs despite harboring high CRBN expression levels. To delineate the molecular mechanisms underlying IMiDs resistance, we first performed genome-wide knockout screening in IMiDs-sensitive MM.1S cells using a CRISPR-Cas9 GeCKOv2 library containing 6 unique sgRNAs against each of 19,050 genes and 4 sgRNAs against each of 1,864 miRNAs. We observed that knockout (KO) of twenty-eight genes and one miRNA were associated with resistance to IMiDs. Of note, all six sgRNAs targeting CRBN were identified as a positive regulator of IMiDs sensitivity, consistent with previous studies. Among these genes, we found that three different sgRNAs targeting TRAF2 were enriched after IMiDs treatment. Therefore, we next individually cloned the sgRNAs of TRAF2 into the Cas9 lentiviral vector, and then re-introduced them into MM.1S cells. Importantly, TRAF2-KO MM.1S cells acquire significant resistance to Pom and Len treatment. To examine whether TRAF2 KO induced IMiDs resistance was CRBN-pathway dependent, we assessed CRBN and its downstream interacting protein levels. TRAF2 KO showed no effect on CRBN expression; moreover, IMiDs can still trigger IKZF1 and IKZF3 degradation, associated with downregulation of IRF4. Taken together, these data suggest that TRAF2 mediates sensitivity to IMiDs in a mechanism independent of CRBN-IKZF1/3 axis. Since TRAF2 is a member of the TNF receptor associated factor (TRAF) protein family required for activation of several signaling pathways including NF-ĸB and JNK, we next examined the biologic impact of TRAF2 KO. Importantly, TRAF2 KO cells show significantly increased processing of precursor p100 to p52 (NF-ĸB2), resulting in hyperactivation of the non-canonical NF-κB pathway; conversely, p52 KO re-sensitizes the cells to IMiDs treatment. The activity of the canonical NF-κB pathway was not similarly altered in TRAF2 KO cells. These results suggest that TRAF2 predominantly activates non-canonical NF-κB pathway, associated with resistance to IMiDs in MM cells. We next examined the relevance of TRAF2 downregulation in the context of the bone marrow (BM) microenvironment. Co-culture of MM cells with either bone marrow stromal cells (BMSCs) or culture supernatants (BM-CS) confers resistance to IMiDs. Importantly, TRAF2 is downregulated in BMSCs and BM-CS, suggesting that IMiDs resistance in the BM microenvironment may be mediated by TRAF2 downregulation induced by soluble factors. Cytokine array assay confirmed detectable TNF-α in BM-CS. Indeed, MM cells treated with TNF-α showed decreased TRAF2 expression, associated with resistance to IMiDs. These data demonstrate that TNF-α secreted by BMSCs induces TRAF2 downregulation, thereby inducing IMiDs resistance. We further analyzed the mechanism of TNF-α-mediated TRAF2 downregulation. Since previous studies have shown that TRAF2 is a substrate of the proteasome, we treated MM cells with TNF-α in the presence and absence of proteasome inhibitor. TRAF2 downregulation by TNF-α was partially abrogated by proteasome inhibitor, associated with accumulation of ubiquitinated TRAF2. These data confirm that TNF-α induces TRAF2 downregulation is due, at least in part to proteasomal degradation. In conclusion, our data show that TRAF2 is a CRBN-independent regulator of IMiDs sensitivity and provide the preclinical rationale for combining IMiDs with inhibitors of non-canonical NF-κB or TNF-α signaling to overcome IMiDs resistance and improve patient outcome. Disclosures Anderson: Sanofi-Aventis: Other: Advisory Board; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder.

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p38 MAPK Controls Sensitivity Towards BH3 Mimetics By Regulating Mcl-1 Expression of Chronic Lymphocytic Leukemia in Hypoxia and Acquired Resistance

Blood ◽

10.1182/blood.v124.21.1947.1947 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1947-1947

Author(s):

Malte F Huelsemann ◽

Michaela Patz ◽

Laura Beckmann ◽

Kerstin Brinkmann ◽

Teresa Otto ◽

...

Keyword(s):

B Cell ◽

P38 Mapk ◽

Cell Lines ◽

Molecular Mechanisms ◽

Acquired Resistance ◽

Bh3 Mimetics ◽

Dna Targeting ◽

Hypoxic Conditions ◽

Response To Chemotherapy ◽

Impact On Survival

Abstract Background: CLL patients frequently suffer relapse after an initially successful chemotherapy. This distinct resistance towards chemotherapy is thought to be caused by microenvironmental stimulation. Within the tumor microenvironment (TME) cells are not only stimulated by well-known external stimuli like CD40 ligand (CD40L) or activation of the B cell receptor (BCR), but are also exposed to hypoxia, which was found in the bone marrow and lymphatic tissue. Despite the known importance of hypoxia in solid tumors, its impact on survival and treatment response in CLL is still poorly understood. Methods: We have established a novel in vitro model for the CLL microenvironment, which considers both the external stimulation by CD40L and the hypoxic oxygen levels (1% O2). Treatment efficacy of different drugs in normoxia (21% O2) and hypoxia were determined by AnnexinV/7-AAD staining and subsequent FACS analysis. The underlying molecular mechanisms were analyzed via qRT-PCR and immunoblot. Furthermore B-cell lines Raji, Ramos and Mec-1 were continuously exposed to increasing concentrations of fludarabine or the BH3 mimetic ABT-737. After establishment of resistance the molecular adaptation was assessed and correlated to the changes induced by hypoxia. Results: Hypoxia is known to protect solid cancers from chemotherapy. In our model we made similar observations for CLL, since sensitivity to the classical DNA-targeting drugs fludarabine and bendamustine was reduced under hypoxic conditions. Interestingly, the tyrosine kinase inhibitor ibrutinib did not benefit from hypoxia either. However, this resistance was overcome by the mitochondria-targeting BH3 mimetics ABT-199 and ABT-737, whose effect was pronounced under hypoxia. We reveal that this effect was caused by an uncoupling of major signaling pathways. Under hypoxic conditions the activity of Akt, ERK1/2 and NFκB was reduced, while p38 MAPK became hyperphosphorylated. Phospho-p38 (pp38) downregulated Mcl-1 levels, which are the main regulator of sensitivity towards BH3 mimetics. Despite the known heterogeneity in between CLL patients this effect was found in most samples analyzed. The functional importance was underlined by the observation that pharmacological inhibition of p38 MAPK could reconstitute Mcl-1 levels and thereby resistance in hypoxia. The relevance of the pp38-Mcl-1 axis for ABT efficacy was emphasized by findings in B-cell lines with acquired resistance. Each ABT-resistant clone of the three tested cell lines induced p38 activity and decreased Mcl-1 levels. In contrast, in the fludarabine-resistant clones the pp38-Mcl-1 axis was not altered. Conclusion: These are the first experiments providing evidence that hypoxia has a crucial impact on survival and response to chemotherapy in CLL. We show that hypoxia renders CLL cells resistant to classical DNA-targeting agents, while the small molecules ABT-199 and ABT-737, which specifically target mitochondria, efficiently eradicate CLL cells within the microenvironment. Furthermore, we identified the pp38-Mcl-1 axis to be a major determinant of sensitivity to these BH3 mimetics, which warrants further evaluation of p38 as a novel biomarker for prediction of sensitivity to BH3 mimetics. Disclosures No relevant conflicts of interest to declare.

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EXTH-50. THE COMPLEXITIES OF FATTY ACID DESATURATION IN GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noab196.689 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi174-vi174

Author(s):

Nicole Oatman ◽

Biplab Dasgupta

Keyword(s):

Fatty Acid ◽

Cell Lines ◽

Acyl Chain ◽

Acquired Resistance ◽

Enzymatic Reaction ◽

Monounsaturated Fatty Acids ◽

Fatty Acid Desaturation ◽

Normal Brain ◽

Drug Induced ◽

Essential Components

Abstract Fatty acid desaturation is an enzymatic reaction in which a double bond is introduced into an acyl chain. Monounsaturated fatty acids (MUFA) are essential components of membrane. The most abundant MUFA-synthesizing enzyme is the delta 9 desaturate called Stearoyl Co-A Desaturase (SCD and SCD5 in humans, and SCD1-4 in mice). SCD desaturates Stearoyl-CoA (C18) and palmitoyl-CoA (C16) to oleoyl-CoA (C18:1) and palmitoyl-CoA (C16:1), respectively. SCD is often upregulated and a therapeutic target in cancer. We made an unexpected discovery that that median expression of SCD is low in glioblastoma relative to normal brain due to hypermethylation and monoallelic co-deletion with the tumor suppressor PTEN in a subset of patients. Cell lines from this subset, expressed nearly undetectable SCD yet they retained residual SCD enzymatic activity. Surprisingly, these lines evolved to survive independent of SCD through unknown mechanisms. On the other hand, cell lines that escaped such genetic and epigenetic alterations expressed higher levels of SCD and were highly dependent on SCD for survival. Finally, we identify that SCD-dependent lines acquire resistance through a previously unknown mechanism that involved drug-induced target (SCD) upregulation by the transcription factor FOSB. Accordingly, FOSB inhibition blunted acquired resistance and extended survival of tumor bearing mice treated with SCD inhibitor. Our findings reveal an intriguing feature of the cancer genome that may be used to stratify PTEN deleted cancer patients for SCD inhibitor therapy. A recent study showed that some cancer cells can use another MUFA-synthesizing enzyme FADS2 to bypass the SCD reaction. However, our data shows that the SCD inhibitor- resistant GBM lines are also FADS2-independent. Our targeted and untargeted metabolomics studies revealed unexpected findings that cannot be explained by conventional wisdom, and may lead to identification of novel lipogenic targets in GBM.

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A Novel Anti-a Proliferation-Inducing Ligand Hapril.01A Monoclonal Antibody Targets Multiple Myeloma Cells in the Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v124.21.2098.2098 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2098-2098

Author(s):

Yu-Tzu Tai ◽

Chirag Acharya ◽

Gang An ◽

Mike Y Zhong ◽

Michele Cea ◽

...

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibody ◽

Bone Marrow ◽

Dna Binding ◽

B Cell ◽

Cell Lines ◽

Plasma Cell ◽

Molecular Mechanisms ◽

Myeloma Cells ◽

Micromolar Range

Abstract A proliferation-inducing ligand (APRIL), a close member of B-cell-activating factor (BAFF) belonging to the TNF superfamily, is mainly produced by bone marrow (BM) accessory cells to stimulates growth and survival of multiple myeloma (MM) cells. Unlike BAFF, APRIL is dispensable in B cell homeostasis but more critical in plasma cell differentiation and survival. It has higher affinity than BAFF (nanomolar vs micromolar range) to B cell maturation antigen (BCMA) (nanomolar vs micromolar range) which expresses at high levels in all patient MM cells. APRIL also binds to a common plasma cell (PC) marker syndecan-1 (CD138) to induce signaling cascade via TACI, the other APRIL receptor in PC. We here characterize molecular mechanisms regulating APRIL activation in the BM microenvironment and further determine whether a novel anti-APRIL monoclonal antibody hAPRIL.01A inhibits its functional sequelae in MM. First, in vitro osteoclast and macrophage cultures were performed by stimulating CD14+ monocytes from MM patient samples with M-CSF/RANKL and M-CSF, respectively. Osteoclasts and macrophages secret significantly higher levels of APRIL than unstimulated CD14+ monocytes and BM stromal cells (BMSC), as confirmed by ELISA and qRT-PCR. All MM cell lines express cell surface BCMA in significantly higher level than TACI (p < 9.06e-15). H929 MM cells (expressing only BCMA, but not TACI), and other MM cell lines were next stimulated with APRIL, in the presence or absence of hAPRIL.01 Ab followed by immunoblotting and TaqMan® Array assays on harvested protein lysates and mRNA. APRIL stimulation consistently activated NF-kB, PI3K/AKT, and ERK1/2 signaling in MM cells. Importantly, NF-kB-DNA binding activities of p65 and p50 (p52, to a less extent), were significantly upregulated as early as 15 minute and sustain to 4h in all MM cell lines after stimulation. Conversely, hAPRIL.01 Ab completely blocked these signaling cascades, consistent with significantly decreased NF-kB-DNA binding activities in BCMA-knock-downed MM cells by shBCMA lentivirus transfection. APRIL further induced pro-survival proteins (Mcl1, Bcl2, BIRC3, XIAP) and MM cell growth-stimulating regulators (CCDN2, CDK4, CDK6, c-myc), which were completely inhibited by hAPRIL.01 Ab. These results correlated with blockade of hAPRIL.01 Ab in APRIL-promoted viability and colony formation of MM cells, in the presence of osteoclasts or macrophages. APRIL also induces adhesion of MM cells to BMSC, which was blocked by hAPRIL.01 Ab. This concurred with hAPRIL.01 Ab-reduced adhesion molecules (CD44, ICAM-1) induced by APRIL. APRIL-increased VEGF-A and PECAM-1 in MM cells was also significantly reduced by this mAb. APRIL-upregulated chemotactic/osteoclast-activating factors (MIP-1α, MIP1β, SDF-1) were also inhibited by this Ab. Other angiogenesis and adhesion/chemoattractant factors, i.e., IL-8, CXCL10, RANTES and MDC (ccl22), were changed in a similar fashion, indicating specific blocking of hAPRIL.01 Ab to APRIL-induced downstream target genes. This mAb further inhibited APRIL-increased viability of plasmacytoid dendritic cells (pDC) and diminished MM cell viability protected by pDC in 3-d cocultures. Finally, hAPRIL.01 specifically overcame APRIL-, but not IL-6, induced protection in lenalidomide- or dexamethasone-treated MM1S and H929 MM cells. These studies confirm a constitutive APRIL activation via BCMA and TACI in promoting malignancies of myeloma cells, supporting a novel therapeutics of hAPRIL.01 Ab to target MM in the BM microenvironment. Disclosures van Eenennaam: BioNovion: Employment. Elsas:BioNovion: Employment. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.

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Low Miki Expression in Myelodysplastic Syndromes Results in Polynuclear Cell Formation through Decondensation of Chromosomes in Prolonged Prometaphase

Blood ◽

10.1182/blood.v126.23.2845.2845 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2845-2845

Author(s):

Akiko Nagamachi ◽

Yuko Ozaki ◽

Hirotaka Matsui ◽

Akinori Kanai ◽

Toshiya Inaba

Keyword(s):

Bone Marrow ◽

Mrna Expression ◽

Cell Lines ◽

Negative Correlation ◽

Hela Cells ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Strong Positive Correlation ◽

Expression Levels ◽

Mrna Expression Levels

Abstract Polynuclear cells (PNCs) are routinely observed in the bone marrow of MDS patients. They are binuclear, trinuclear or even multinuclear cells with or without micronuclei, the underlying molecular mechanisms for the production of which are largely unknown. Because loss of the long arm of chromosome 7 (7q-) was reported to be associated with the presence of a higher frequency of PNCs, gene(s) preventing bone marrow cells from carrying such nuclear abnormalities may be located at 7q. We previously identified three candidate anti-myeloid tumor suppressor genes, namely Samd9, Samd9L and Miki, from the microdeletion in the 7q21 band frequently detected in JMML patients. SAMD9L-deficient mice develop MDS resembling human diseases associated with 7q-, most likely through enhancement of cytokine signals (Nagamachi et al., Cancer Cell 2013). Miki (mitotic kinetics regulator) translocates from the Golgi apparatus to mitotic centrosomes coincident with the disappearance of the Golgi body after poly-ADP-ribosylation (PARsylation). Miki is indispensable for centrosome maturation [the rapid increase of pericentriolar materials (PCM) during prophase and prometaphase], which is required for the production of robust mitotic spindles to move chromosomes promptly (Ozaki et al., Mol. Cell 2012). Consequently, as observed by time-lapse imaging of HeLa cells expressing histone H2A-GFP, downregulation of Miki by siRNA markedly prolonged the duration of prometaphase to more than several hours (normally around 15 minutes). Chromosomes were scarcely able to align and cells exited from prometaphase either by cell death or by decondensation of each chromosome. In the latter, cells with decondensed chromosomes then fused with one another within 30 minutes to form cells with relatively large nuclei, resulting in PNCs containing various sizes of nuclei including micronuclei. Indeed, reduction of Miki in HeLa cells by siRNA increased the frequency of PNCs from less than 0.5% to 4.5%. To test whether the chaotic chromosome decondensation in prometaphase causes the accumulation of PNCs observed in MDS, we initially used five cell lines derived from MDS associated with 7q-. PARsylated Miki was barely detectable in these cell lines and we found more cells at prometaphase than at metaphase (the ratio of prometa:meta in the lines ranged from 1.7:1 to 5.7:1). In contrast, in seven cell lines expressing PARsylated Miki at high levels, mitotic cells in prometaphase were found less frequently or at roughly the same frequency as those in metaphase (prometa:meta ratio 0.6:1 to 1.3:1). PNCs in five cell lines harboring 7q-were also more frequent (5.9 - 10.2%) than in the seven cell lines expressing high PARsylated Miki (0.8 - 2.4%). In addition, when we reduced Miki expression levels by shRNA in K562 cells, which express PARsylated Miki at high levels, the prometa:meta ratio increased from 1.1:1 to 3.8:1 and PNCs increased from 0.8% to 8.5%. This suggests that, as in HeLa cells, low expression levels of Miki cause prolongation of prometaphase and increase PNCs in blood cells. Fresh bone marrow preparations from 37 patients with MDS were examined to determine whether Miki mRNA-expression levels influence the prometaphase:metaphase ratio and the frequency of PNCs. We found a strong negative correlation (R=-0.59, p<0.01) between Miki mRNA expression levels in mononuclear cells of bone marrow samples and the prometa:meta ratio. We also found a moderate negative correlation (R=-0.4, p<0.05) between PNC frequencies and Miki mRNA expression levels. In addition, there was a strong positive correlation between prometa:meta ratios and PNC frequencies (R=-0.56, P<0.01). In conclusion, lack of one allele of the Miki gene due to 7q-reduces PARsylated Miki, resulting in the increase of PNCs through decondensation of chromosomes in prolonged prometaphase. This may contribute to poor outcome of MDS associated with 7q-through increased chromosome instability. Disclosures No relevant conflicts of interest to declare.

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