The Direct Interactions with Bone Marrow Microenvironment Confer Resistance to the Inhibition of Oxidative Phosphorylation in AML

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-11
Author(s):  
Yoko Tabe ◽  
Kaori Saito ◽  
Kotoko Yamatani ◽  
Haeun Yang ◽  
Rodrigo Jacamo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lipid Metabolism ◽  
Mouse Models ◽  
Stromal Cells ◽  
Research Funding ◽  
Ldl Receptor ◽  
Leukemia Cells ◽  
Related Protein ◽  
Resistant Mouse ◽  
Argininosuccinate Synthase

Acute myeloid leukemia (AML) cells are highly dependent on oxidative phosphorylation (OxPhos) for survival and continually adapt to the bone marrow (BM) microenvironment. We investigated how the BM microenvironment impacts the response to energy-depriving OxPhos inhibition in AML using a novel complex I OxPhos inhibitor (OxPhosi), IACS-010759. We have reported that OxPhosi-resistant primary AML samples demonstrated higher baseline transcription of genes related to cell adhesion, integrin and/or Rho GTPase family genes that modulate intracellular actin dynamics. (Yang et al. ASH 2019) In this study, we performed Cap Analysis of Gene Expression (CAGE) transcriptome analyses using IACS-010759-sensitive and -resistant AML PDXs. CAGE identifies and quantifies the 5' ends of capped mRNA transcripts (= transcription start sites) and allows investigating promoter structures necessary for gene expression. Primary AML cells from 9 AML PDXs were injected into irradiated NSG mice, which were randomized upon documented engraftment to receive IACS-010759 or vehicle (n = 3/group). The antileukemia efficacy of the treatment was monitored by serial measurements of circulating AML cells. Of the 9 models tested, we defined 4 PDXs as sensitive and 5 as resistant to OxPhos inhibitor therapy. In the resistant models, CAGE analysis of OxPhosi-induced changes (comparing pretreatment with posttreatment) identified upregulation of 77 promoters and downregulation of 207 promoters (log 2-fold change > 3.0, FDR < 0.05, EdgeR), including increased promoter expression (>3.0 fold) of genes associated with adhesion (CCR8,ADGRB2, LAG3, BMF, ATN1, PLXDC1), migration (CCR8, NKX3-2, TMEM123, IGLV7-43, FAM171A1, LBX2, TRAV21, PPP2R5C, BMF, PLXDC1), and actin cytoskeleton dynamics (FAM171A1, BMF, BEST1, PLXDC1). Of note, the 6 adhesion-associated promoters that were upregulated by OxPhosi in 5 of the OxPhosi-resistant mouse models were unchanged or downregulated in the 4 OxPhosi-sensitive models. We then used DEGseq, an R package for identifying differentially expressed genes, to identify promoters whose expression was different between OxPhosi-treated and vehicle-treated groups in the OxPhosi-resistant mouse models. DEGseq detected consistent changes of 214 upregulated and 626 downregulated promoters with OxPhosi treatment in all 5 mouse models. KEGG pathway enrichment analysis was performed with these consistently changed genes and revealed that OxPhos inhibitor treatment significantly upregulated the transcripts of cell adhesion pathway. We then confirmed that BM derived mesenchymal stem cells (MSC) protected OxPhosi-sensitive OCI-AML3 cells; the IC50 of IACS-010759 under MSC coculture was 80-fold higher than in monoculture conditions (IC50; 0.04 nM in monoculture vs. 3.25 nM in coculture), and IACS-010759 (10nM) induced 55% reduction of viable cells in coculture condition as compared to 70% reduction in monoculture. We further observed that OCI-AML3 cells adhered to MSCs were more profoundly protected from OxPhosi induced apoptosis than nonadherent cells. These results indicate that BM stromal cells, in particular those in direct contact with leukemia cells, play a key role in the microenvironment-mediated protection of AML cells from metabolic stress caused by OxPhos blockade. We further observed promoter upregulation of ASS1, coding Argininosuccinate Synthase 1 and of LRP1, coding LDL Receptor Related Protein 1. Argininosuccinate Synthase 1 is an epigenetically regulated key enzyme in the biosynthesis of arginine and energy starvation that induces adaptive transcriptional upregulation of ASS1. LDL Receptor Related Protein 1 plays a major role in lipid metabolism and has been reported to be responsible for hemin-induced autophagy in leukemia cells. These might contribute to intrinsic AML resistance to OxPhosi via activation of compensatory metabolic pathways, amino acid metabolism and lipid metabolism. Taken together, our data highlight the importance of direct interaction with BM stromal cells as well as complementally modification of amino acid- and lipid metabolism for the resistance of AML cells to OxPhos inhibition. While the mechanisms of stroma-leukemia interactions are likely complex, reducing the adhesion of AML cells to nurturing stromal cells ameliorates the resistance to the metabolic and energetic consequences of OxPhos inhibition. Disclosures Andreeff: Amgen: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding. Konopleva:Rafael Pharmaceutical: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Sanofi: Research Funding; AstraZeneca: Research Funding; Cellectis: Research Funding; AbbVie: Consultancy, Research Funding; Ablynx: Research Funding; Agios: Research Funding; Ascentage: Research Funding; Eli Lilly: Research Funding; Forty-Seven: Consultancy, Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Amgen: Consultancy; F. Hoffmann La-Roche: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Kisoji: Consultancy; Calithera: Research Funding.

In Silico Prediction of Novel Drug Combinations to Combat Bortezomib-Resistant Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1344-1344
Author(s):  
Holly A. F. Stessman ◽  
Tian Xia ◽  
Aatif Mansoor ◽  
Raamesh Deshpande ◽  
Linda B. Baughn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
In Silico ◽  
Research Funding ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Mouse Cell ◽  
Resistant Mouse ◽  
Novel Therapeutic

Abstract Abstract 1344 Bortezomib/VELCADE® (Bz) is a proteasome inhibitor that has been used successfully in the treatment of multiple myeloma (MM) patients. However, acquired resistance to Bz is an emerging problem. Thus, there is a need for novel therapeutic combinations that enhance Bz sensitivity or re-sensitize Bz resistant MM cells to Bz. The Connectivity Map (CMAP; Broad Institute) database contains treatment-induced transcriptional signatures from 1,309 bioactive compounds in 4 human cancer cell lines. An input signature can be used to query the database for correlated drug signatures, a technique that has been used previously to identify drugs that combat chemoresistance in cancer (Wei, et al. Cancer Cell (2006) 10:331). In this study we used in silico bioinformatic screening of gene expression profiles from isogenic pairs of Bz sensitive and resistant mouse cell lines derived from the iMycCα/Bcl-xL mouse model of plasma cell malignancy to identify compounds that combat Bz resistance. We established Bz-induced kinetic gene expression profiles (GEPs) in 3 pairs of Bz sensitive and resistant mouse cell lines over the course of 24 hours. GEPs were collected in the absence of large-scale cell death. The 16 and 24 hour time points were averaged and compared between each Bz sensitive and resistant pair. Genes in the sensitive cell line with a fold change greater than 2, relative to the resistant line, were given the binary distinction of “up” or “down” depending on the direction of change. Genes that met these criteria were assembled into signatures, and then used as inputs for CMAP queries to identify compounds that induce similar transcriptional responses. In all pairs, treatment of the Bz sensitive line correlated with GEPs of drugs that target the proteasome, NF-κB, HSP90 and microtubules, as indicated by positive connectivity scores. However eight compounds, all classified as Topoisomerase (Topo) I and/or II inhibitors, were negatively correlated to our input signature. A negative connectivity score could have two interpretations: (1) this could indicate simply that Topos are upregulated by Bz treatment in Bz sensitive lines, which has been previously reported (Congdan, et al. Biochem. Pharmacol. (2008) 74: 883); or (2) this score could be interpreted as Topos are inhibited in Bz resistant cells upon Bz treatment. This led us to ask whether Topo inhibitors could target Bz resistant MM cells and re-sensitize them to Bz. Indeed, we found that multiple Topo inhibitors were significantly more active against Bz resistant cells as single agents and restored sensitivity to Bz when combined with Bz as a cocktail regimen. This work demonstrates the potential of this in silico bioinformatic approach for identifying novel therapeutic combinations that overcome Bz resistance in MM. Furthermore, it identifies Topo inhibitors – drugs that are already approved for clinical use – as agents that may have utility in combating Bz resistance in refractory MM patients. Disclosures: Stessman: Millennium: The Takeda Oncology Company: Research Funding. Van Ness:Millennium: The Takeda Oncology Company: Research Funding.

scholarly journals Low-Dose Ionizing Radiation Modulates Expression of Gpam in Immortalized EBV Infected B Cells through miRNA Network

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4829-4829
Author(s):  
Yoko Tabe ◽  
Yasuhito Hatanaka ◽  
Mayumi Nakashiro ◽  
Kazumasa Sekihara ◽  
Shinichi Yamamoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Ionizing Radiation ◽  
Mirna Expression ◽  
Stromal Cells ◽  
Research Funding ◽  
Low Dose ◽  
Binding Protein ◽  
B Cell Lymphoma ◽  
Stromal Microenvironment

Abstract Low-dose ionizing radiation (LDIR, ≤ 0.1 Gy) which is typically associated with therapeutic and diagnostic radiological modalities, is known to induce remodeling of the stromal microenvironment. Whereas the biologic responses to LDIR are commonly described as a stress response, the carcinogenic potential of this environmental stressor remains unknown. Recently, ionizing radiation (IR)-induced alterations of miRNA expression that play a fundamental role in cell signaling events, have been demonstrated in various cell types (Marsit, Cancer Res. 2006). To assess a potential determinant influencing of LDIR induced miRNA alterations in pre-malignant cells in a microenvironment, we utilized immortalized pre-malignant Epstein-Barr virus infected-B (EBV-B) cells which are continuously proliferating in circulation or in lymph nodes. The LDIR system (0.1 Gy, 4MV X ray from a LINAC) was utilized in the in vitro co-culture of EBV-B and mesenchymal stromal cells (MSC) to mimic the lymph node stromal microenvironment. We confirmed that MSC protected co-cultured EBV-B cells from spontaneous apoptosis and caused accumulation of EBV-B cells in the G0/G1 phase (EBV-B; monoculture vs coculture with MSC; Sub G1% 42.0±2.4 vs 34.1±0.9 p< 0.01, G0/G1 % 42.6±2.1 vs 47.6±2.0, p<0.05). To identify LDIR-induced modulation of specific miRNAs in EBV-B cells, a high-confidence list of 44 known LDIR-associated miRNAs from three separate screening studies of lymphoblastic cells was assembled (Cha et al., Oncol Rep.2009, Chaudhry et al., J Biomed Sci. 2010, Lhakhang et al., Comp Funct Genomics. 2012). From these miRNA, we focused on four miRNAs let-7a, miR-16, miR-19b and miR-21 that have been listed in more than two of the screens. Our studies showed that LDIR upregulated let-7a and miR-16 levels in irradiated mono-cultured EBV-B, but on the contrary downregulated all tested miRNAs in MSC co-cultured EBV-B cells (at 24-hour). We next investigated alterations of EBV-B gene expression by LDIR using the DNA microarray (Affymetrix). In mono-cultured EBV-B cells, cDNA microarray analysis detected upregulation of TGFB1 mRNA, and downregulation of the genes encoding a lipid biosynthesis enzyme glycerol-3-phosphate acyltransferase (GPAM), a growth factor amphiregulin (AREG), cell survival-/growth-related factors Rho-GTPase effector gene folmin-1 (FMN1), chemotaxis-inducing chemokine gene IL-8 and B-cell lymphoma 2 gene BCL2, all of which were confirmed by qRT-PCR. Downregulation of the GPAM protein was further shown on the protein level by western blot, and the ontology analysis demonstrated that LDIR caused the TGFbeta-dependent induction of the epithelial-mesenchymal transition (EMT) pathway in mono-cultured EBV-B cells. In contrast to the mono-culture condition, the GPAM mRNA and protein expression were upregulated in MSC co-cultured EBV-B cells. To determine the potential targets of LDIR-altered miRNAs, we next identified 79 genes that are commonly targeted by let-7a, miR-16, miR-19b, or miR-21 from a microrna.org database, and found that the expression changes of GPAM mRNA and protein were strikingly matched with miRNA profiling. Partial concordant changes of DNAJ(HSP40)A2 coding heat shock protein (HSP) 40 homolog, a cochaperone of HSP70s, CPEB3 coding RNA binding protein cytoplasmic polyadenylation element binding protein 3 (CEBP3) and its transcriptional target GLUR2 (glutamate receptor subunit), a receptor of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), were also observed in the microarray results. Since GPAM is a lipid-related metabolism gene which is known to be regulated by miR-27b and to negatively impact survival of breast cancer cells we next investigated the expression changes of miR-27b after LDIR in EBV-B cells. As expected, LDIR upregulated miR-27b in mono-cultured EBV-B cells, and downregulated in co-cultured EBV-B cells with MSC. Intriguingly, no significant changes of GPAM mRNA as well as tested miRNA expression was observed by 1Gy irradiation. In summary, we demonstrated that LDIR directly modulates EBV-B cells gene expression. Importantly, LDIR additionally impacts EBV-B cells indirectly through miRNA modulation by the neighboring stromal cells. Disclosures Konopleva: Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

scholarly journals Functional and Genomic Characterization of the Interaction between Acute Lymphoblastic Leukemia Cells and the Microenvironment Identifies Pathways for Therapeutic Intervention

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1550-1550
Author(s):  
Hiroki Yoshihara ◽  
Michelle L. Churchman ◽  
Jennifer L. Peters ◽  
David B. Finkelstein ◽  
Elisabeth M. Paietta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Time Lapse ◽  
Leukemia Cells ◽  
Adherent Cells ◽  
Bone Marrow Mscs ◽  
Upstream Regulator

Abstract Introduction Residence and interaction with a specialized bone marrow microenvironment is important for normal hematopoietic stem cells and for initiation and progression of myeloid malignancies, but the role of the microenvironment in propagation and therapeutic response of acute lymphoblastic leukemia (ALL) is not well known. Prior work has identified the efficacy of inhibiting FAK signaling, which is deregulated by IKZF1 alterations resulting in induction of THY1-Integrin alpha 5 adhesion in Ph-positive (Ph+) ALL. Here, we hypothesized that this mechanism may be more broadly important in ALL. We applied a systematic integrated genomic/imaging/functional approach to define the nature of interaction and identify changes in leukemic cells upon interaction that may be targetable. Materials and methods Time-lapse confocal imaging was performed to examine how leukemia cells migrate and adhere to mesenchymal stem cells (MSCs). NALM6 (DUX4/ERG), MHH-CALL2 (hypodiploid), 697 (TCF3-PBX1), Reh (ETV6-RUNX1) and SUP-B15 (Ph+) cell lines were cultured with immortalized human bone marrow MSCs transduced with telomerase reverse transcriptase (hTERT) (Mihara, Br J Haematol. 2003;120:846). For RNA-sequencing, non-adherent cell line cells were collected after two days of coculture with hTERT while adherent cells were trypsinized and collected. Both samples were sorted for CD19 positive population. Fresh primary ALL samples were cultured on bone marrow MSCs derived from patients with no hematological disease and collected with the same procedure for RT-PCR. Multicolor immunofluorescence imaging was utilized to observe expression of multiple molecules involved in adherence. Results Time-lapse imaging showed that leukemia cells have a dynamic interaction with MSC monolayers, with temporary adherence, accompanied by dynamic change in their shape. NALM6 cells adherent to MSCs reduced cell cycling, with an increase in the ratio of G0/G1 cells (26.7% to 48.0%) and decrease in S phase (60.7 to 41.8%). Analysis of gene expression showed 138 upregulated genes (log2FC >2 and FDR <0.05) in adherent cells which were common in all five cell lines, with striking upregulation leading to gene expression associated with gene ontology of extracellular matrix organization and collagen fibril organization. Representative genes were validated in adherent NALM6 cells by immunoblotting (FN1, TIMP1, and LGALS1). Pathway analysis showed that transforming growth factor beta 1 (TGFB1) was the top ranked upstream regulator (p-value of overlap 5.26E-35) in that 44 of 65 genes had measurement direction consistent with activation of TGFB1 signaling. In addition, other upstream regulators that may be involved were beta-estradiol, fibroblast growth factor 2, and tumor necrosis factor. Adhesion of leukemia cells to stroma may induce integrin expression and downstream signaling. Our transcriptomic analysis showed that integrins (A5, B1, A3 and B5) and caveolin 1 (CAV1), a main component of the caveolae plasma membranes, are highly transcribed in adherent leukemia cells. As hTERT cells showed unexpectedly low expression of THY1, a common MSC marker, we utilized primary bone marrow MSC for subsequent analysis. Immunoblotting assay showed enhanced expression of CAV1 in all cell lines adhered to MSCs. Multicolor immunofluorescence imaging demonstrated CAV1 and ITGB1 expression on leukemia cells that localized adjacent to stromal cells, which confirms that these molecules were upregulated upon adhesion to MSCs. Moreover, primary ALL cells showed remarkable upregulation of CAV1, ITGB1, ITGA3, and ITGB5 when the cells were adherent to MSCs. Conclusions Our results demonstrate that ALL cells dynamically interact with microenvironment cells, inducing changes in cell morphology, cell cycling, and adhesion, which may facilitate altered responsiveness to therapy. Transcriptional results suggest that TGFβ signaling is an upstream regulator after cell adhesion to MSCs. While integrins and CAV1 mediate the signaling, these pathway and molecules will be candidates for exploring inhibitors of signaling, which may affect their interaction and make them novel therapeutic targets. Figure. Figure. Disclosures Mullighan: Loxo Oncology: Research Funding; Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau.

Cage Transcriptome Analysis Reveals BCL2A1 Upregulation in FLT3-ITD/D835 Dual Mutated AML Cells Harboring Complex Co-Mutations

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1264-1264
Author(s):  
Kotoko Yamatani ◽  
Tomohiko Ai ◽  
Kaori Saito ◽  
Haeun Yang ◽  
Koya Suzuki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tyrosine Kinase ◽  
Board Of Directors ◽  
Research Funding ◽  
Leukemia Cells ◽  
Genetic Mutations ◽  
Advisory Committees ◽  
Protein Levels ◽  
Significant Difference ◽  
Equity Ownership

Genetic mutations in FLT3 (fms-like tyrosine kinase-3) play an important role in the pathogenesis of acute myeloid leukemia (AML). FLT3 internal tandem duplications (FLT3-ITD) occur in approximately 25% of all AML cases and various tyrosine kinase inhibitors (TKIs) targeting FLT3-ITD such as quizartinib, crenolanib, and gilteritinib have been developed. Although these selective FLT3 inhibitors were thought to be promising, their effects turned out to be temporary due to the rapid development of resistance associated with clonal switching. Acquired FLT3 point mutations at D835 in the activation loop of tyrosine kinase domain are often accountable for clonal switching at least for Type II TKIs. In addition, adjunct mutations in other genes are also found to be associated with TKI resistance. To investigate the underlying molecular mechanism of this secondary, mutation-driven acquired resistance, we first analyzed co-occurring mutations in the leukemia cells obtained from 26 AML patients with FLT3-ITD (n=14) or FLT3-ITD/D835 dual (n=12) mutations, and performed cap analysis of gene expression (CAGE) sequencing, which identifies and quantifies the 5' ends of capped mRNA transcripts (transcription start sites) and allows investigating promoter structures necessary for gene expression. Patients with FLT3-ITD/D835 harbored a higher number of co-mutations such as ASXL1 and RUNX1 compared to AML with FLT3-ITD (FLT3-ITD/D835: 2.83 ± 0.52, FLT3-ITD: 0.49 ± 0.13, p<0.0001). Intriguingly, CAGE detected significantly higher expression of the anti-apoptotic Bcl-2 family genes BCL2 and BCL2A1 in FLT3-ITD/D835 compared to FLT3-ITD mutant primary samples. Specifically, the CAGE peak of BCL2 was highest in samples with FLT3-ITD/D835 alone (p<0.01), while the CAGE peak of BCLA1 was highest in samples with FLT3-ITD/D835 and co-mutations compared with the other samples (p=0.01). To recapitulate the observations obtained with primary human AML samples, we generated MV4;11 cells with acquired FLT3-ITD/D835 mutations (MV4;11-QR cells) by culturing FLT3-ITD MV4;11 leukemia cells in the presence of quizartinib (1.5 nM), a selective FLT3 inhibitor, for 6 months. While quizartinib (0.2 nM) suppressed the proliferation of 50% of the parental MV4;11 at 72 hours, much higher concentrations of quizartinib (10 nM) was required to suppress the proliferation of MV4;11-QR cells. Quantitative RT-PCR and immunoblot analysis revealed that MV4;11-QR cells expressed higher transcript and protein levels of BCL2A1 than MV4;11 parental cells, while BCL2 levels were similar in both cells and MCL1 and BCLxL expression were lower in the MV4;11-QR than in the parental cells. Next, to investigate the molecular properties of AML cells bearing FLT3-ITD or FLT3-ITD/D835 without other co-mutations, we created Ba/F3 cells stably expressing FLT3-ITD or FLT3-ITD/D835. Of notes, the FLT3-ITD/D835 Ba/F3 cells expressed markedly higher BCL2 transcript and protein levels with lower expression of BCLxL than in FLT3-ITD Ba/F3 cells. No significant difference of MCL1 expression was observed. The sensitivity to quizartinib was massively decreased in the FLT3-ITD/D835 Ba/F3 cells (IC50: FLT3-ITD/D835 >1000nM vs. FLT3-ITD, 0.8nM, at 48h). Finally, we examined the efficacy of the BCL-2 specific inhibitor venetoclax in FLT3-ITD/D835 dual mutated cells with or without upregulation of BCL2 or BCL2A1, the latter shown to confer resistance to venetoclax by sequestering released BIM (Esteve-Arenys, Oncogene. 2018). As expected, venetoclax caused more profound cell growth inhibition and apoptosis induction in BCL2 upregulated FLT3-ITD/D835 Ba/F3 compared to FLT3-ITD Ba/F3 cells (IC50: FLT3-ITD/D835 301nM vs. FLT3-ITD >1000 nM, 96 h). However, FLT3-ITD/D835 bearing MV4;11-QR cells with upregulated BCL2A1 were less sensitive to venetoclax than MV4;11 parental cells (IC50: MV4;11-QR, 149nM vs. MV4;11, 33 nM, 72 h). In conclusion, these results demonstrate that acquisition of D835 mutation in FLT3-ITD mutated AML is often accompanied with multiple co-occurring genetic mutations, and depends on anti-apoptotic BCL-2 associated pro-survival mechanisms. BCL2A1 upregulation might be involved in pathogenesis of acquired drug resistance of FLT3-ITD/D835 dual mutant AML cells, and is a promising new target in FLT3-ITD/D835 refractory AML with complex mutations. Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Shah:Bristol-Myers Squibb: Research Funding. Konopleva:Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding. Andreeff:Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees.

The Actin Binding Protein Plastin-3 Is Involved in the Pathogenesis of Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1662-1662
Author(s):  
Arne Velthaus ◽  
Kerstin Cornils ◽  
Saskia Grüb ◽  
Hauke Stamm ◽  
Daniel Wicklein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Binding Protein ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Mutational Status ◽  
Acute Myeloid

Abstract Leukemia-initiating cells reside within the bone marrow (BM) in specialized niches where they undergo complex interactions with their surrounding stromal cells. In order to identify genes being implicated in the interaction of acute myeloid leukemia (AML) cells and stromal cells, we performed co-cultures of primary AML cells with primary endothelial cells and osteoblasts. The gene expression of co-cultured AML blasts was compared to AML cells grown without adherent cells using microarray analysis. Amongst those genes being dysregulated upon co-culture was the actin binding protein plastin-3 (PLS3). Further RT-qPCR analysis revealed an endogenous PLS3 expression in about 50% of BM samples from AML patients (n=25). In contrast, expression of PLS3 was only detected in 2 of 12 analyzed AML cell lines with Kasumi-1 showing strong and THP-1 showing only weak expression. Therefore, functional analysis of PLS3 in AML was studied using shRNA knockdown and overexpression of PLS3 in Kasumi-1 cells. We could show that PLS3 has an impact on the colony formation capacity of AML cells in vitro as the knockdown resulted in significantly reduced colony numbers while increased colony growth was observed in the Kasumi-1 cells overexpressing PLS3 (p<0.001 and p<0.001, respectively). To investigate the role of PLS3 in vivo, NSG mice were transplanted with the PLS3 knockdown Kasumi-1 cells. Compared to mice transplanted with Kasumi-1 cells transduced with a vector carrying a scrambled shRNA, the PLS3 knockdown mice survived significantly longer (median survival time 64 vs. 110 days, respectively; p<0.001; n=9 mice per group). Finally, we investigated whether the expression of PLS3 was associated with AML patients' outcome using published microarray-based gene expression data (Verhaak et al, Haematologica 2009;94). Clinical data of 290 AML patients were available. Based on the mean gene expression value, the patient cohort was divided into high vs low PLS3 expressors. The overall survival was analyzed in a multivariate Cox proportional hazards model including PLS3 gene expression and the baseline parameters age, karyotype and FLT3 mutational status. After a stepwise removal of insignificant terms, the patient's age and a high PLS3 expression remained as independent prognostic survival markers (for PLS3: HR 1.58 (CI 1.05 - 2.37) and for age: HR 1.01 (CI 1.00 - 1.03)). In conclusion, our results identify the actin binding protein PLS3 as potential novel therapeutic target in AML. Disclosures Stamm: Astellas: Other: Travel, Accommodation, Expenses. Heuser:BerGenBio: Research Funding; Tetralogic: Research Funding; Novartis: Consultancy, Research Funding; Celgene: Honoraria; Bayer Pharma AG: Research Funding; Pfizer: Research Funding; Karyopharm Therapeutics Inc: Research Funding. Fiedler:Kolltan: Research Funding; Ariad/Incyte: Consultancy; Novartis: Consultancy; Gilead: Other: Travel; Teva: Other: Travel; GSO: Other: Travel; Pfizer: Research Funding; Amgen: Consultancy, Other: Travel, Patents & Royalties, Research Funding. Wellbrock:Astellas: Other: Travel, Accommodation, Expenses.

scholarly journals Analysis of Lipid-Associated Gene Expression in Mouse Endotoxin Model

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Charles Price ◽  
Takashi Hato
Keyword(s):  
Gene Expression ◽  
Lipid Metabolism ◽  
Low Dose ◽  
Ldl Receptor ◽  
Kidney Injury ◽  
Clinical Problem ◽  
Renal Tissue ◽  
Receptor Expression ◽  
High Dose ◽  
Endotoxemia Model

Background/Objective:   Sepsis-induced kidney injury is a major clinical problem and is an independent risk factor for mortality. We and others have reported that renal tissue metabolism is profoundly altered in the septic milieu. However, whether such metabolic shift is an adaptive response or pathologic process remains unclear. For instance, decreased lipid metabolism in the renal peroxisomes could limit the genesis of deleterious reactive oxygens species whereas the lack of lipid usage in peroxisomes could contribute to energy depletion. Accordingly, here we examined gene expression changes involved in lipid metabolism using mouse models of endotoxemia. Specifically, we compared gene expression changes between injurious high-dose endotoxemia and protective low-dose endotoxin preconditioning models with the goal of identifying favorable reprogramming in lipid metabolism.     Methods:  To induce protective preconditioning, mice were subjected to low-dose endotoxin followed 24 hours later by high-dose endotoxin. Non-preconditioned mice were subjected to a single high-dose endotoxin. Renal tissues were processed and single-cell RNA-sequencing was performed. Tissue metabolomic data was also obtained.     Results:   We found that renal lipid metabolism is deranged in the non-preconditioned endotoxemia model. Plin2, a marker for lipid accumulation, was significantly upregulated in the proximal tubules of non-preconditioned animals. In contrast, Plin2 expression was preserved in the preconditioned animals, suggesting that altered Plin2 expression is a maladaptive response. Similarly, Pdzk1 and Lrp2, genes involved in HDL/LDL receptor expression, were concurrently downregulated in the injurious model, whereas their expression levels were preserved in the protective model. The stable expression of these two genes may be important for maintaining metabolic capacity and anti-inflammatory effects observed in the preconditioned state. These findings were further supported by our tissue metabolomics analysis.     Conclusion/Impact:   Our data indicate that aberrant lipid metabolism is a prominent feature of endotoxin-induced kidney injury. The use of protective preconditioning served as a platform to identify several candidate genes that could be studied further for the development of biomarkers and precise intervention. 

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