scholarly journals Transcription Factor RUNX1 Regulates Factor FXIIIA Subunit (F13A1) Expression in Megakaryocytic Cells and Platelet F13A1 Expression is Downregulated in RUNX1 Haplodeficiency

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 25-26
Author(s):  
Natthapol Songdej ◽  
Fabiola Del carpio-cano ◽  
Guangfen Mao ◽  
Jeremy Wurtzel ◽  
Lawrence Goldfinger ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Board Of Directors ◽  
Binding Sites ◽  
Research Funding ◽  
Promoter Activity ◽  
Platelet Dysfunction ◽  
Clot Retraction ◽  
Advisory Committees ◽  
Hel Cells ◽  
Runx1 Mutation

Plasma Factor XIII, a transglutaminase, is a complex of two A (gene F13A1) chains with catalytic activity and two B chains (gene F13B). FXIII induces clot stabilization and retraction via cross-linking of fibrin monomers and other proteins. Factor XIII deficiency in plasma causes a major bleeding disorder with impaired clot retraction. FXIIIA is synthesized in megakaryocytes, and is abundant in platelets. It is expressed on activated platelets and cross-links proteins on platelet surface. Little is known regarding the regulation of F13A1 in megakaryocytes and platelets. RUNX1 is a major hematopoietic transcription factor and regulates expression of numerous genes in megakaryocytes and platelets. Patients with RUNX1 haplodeficiency (RHD) with heterozygous mutations have thrombocytopenia, platelet dysfunction with impaired responses to activation, and predisposition to myeloid malignancies. Platelet expression profiling using Affymetrix microarrays of a patient (P1) with a heterozygous RUNX1 mutation (c.969-323G>T) revealed decreased F13A expression, which encodes for subunit A of FXIII: fold-change 0.30; p=0.006; patient profiled twice 10 months apart and compared to 6 healthy subjects. These findings were confirmed by qPCR With RT-PCR platelet F13A mRNA expression was decreased to ~ 20% of mean expression in 4 subjects. Platelet F13A protein expression was decreased by ~50% as assessed by corrected total cellular fluorescence imaging. Platelet F13A mRNA expression was also decreased (to less than 20% of expression in 5 controls) in two siblings (P2 and P3 ages 8 and 3 years) from an unrelated family with a heterozygous RUNX1 mutation 508+1G>A). Plasma FXIII was normal in the patient P1. We studied the regulation of F13A by RUNX1 in human erythroleukemia (HEL) cells treated with phorbol myristate acetate (PMA) to induce megakaryocytic transformation. In silico analyses of the 5' upstream region of F13A showed 7 RUNX1 consensus sites within 545bp from ATG. Chromatin immunoprecipitation studies performed using HEL cells and RUNX1 antibody showed that that RUNX1 binds in the region encompassing site 4 and sites 5-7. In parallel, electrophoretic mobility shift assay (EMSA) studies showed binding of nuclear protein to labeled DNA probes with sites 1, 4, 5, 6, and 7. RUNX1 antibody produced a "supershift" with addition suggesting RUNX1 binding. Site-directed mutagenesis of the RUNX1 binding sites in F13A1 promoter decreased promoter activity in luciferase reporter studies in HEL cells, providing evidence that the binding sites are functional. Further, overexpression of RUNX1 in HEL cells increased F13A1 promoter activity and protein while siRNA RUNX1 knockdown reduced F13A1 protein. We assessed clot retraction over 90 min at 37oC in the patient P1 and his daughter (also with RUNX1 mutation) in citrated whole blood following addition of tissue factor (1 pM) and CaCl2 (1 mM), and in washed platelets resuspended in platelet poor plasma from a healthy subject supplemented with added fibrinogen (0.5 mg/mL) and stimulated with 0.5 U/mL thrombin in the presence of CaCl2. These preliminary studies suggested that clot retraction was slower in the patient P1 compared to the controls, particularly in studies with platelet suspensions. Conclusions: Overall, these studies provide the first evidence that hematopoietic transcription factor RUNX1 regulates expression of F13A1 in megakaryocytic cells and that platelet expression of F13A1 is decreased in platelets from patients with RUNX1 haplodeficiency. The decreased expression of F13A1 in RHD may contribute to the platelet dysfunction in RHD. These findings are particularly interesting because they reflect regulation of a coagulation protein (FXIIIA) by hematopoietic transcription factor RUNX1. Disclosures Lambert: Dova: Consultancy, Membership on an entity's Board of Directors or advisory committees; Principia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shionogi: Consultancy; Educational Concepts in Medicine: Consultancy; Octapharma: Consultancy, Research Funding; Bayer: Consultancy; Platelet Disorder Support Association (PDSA): Consultancy; 22qSociety: Consultancy; ITP Australia: Consultancy; CdLS Foundation: Consultancy; RDMD ITP study: Consultancy; Sysmex: Research Funding; AstraZeneca: Research Funding; Argenix: Consultancy; ClinGen: Honoraria.

scholarly journals RAB31-Mediated Endosomal Trafficking Is Defective in RUNX1 Haplodeficiency

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 519-519 ◽  
Author(s):  
Gauthami S. Jalagadugula ◽  
Guangfen Mao ◽  
Lawrence E. Goldfinger ◽  
Jeremy Wurtzel ◽  
Michele P. Lambert ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Promoter Activity ◽  
Healthy Controls ◽  
Advisory Committees ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Hel Cells ◽  
Runx1 Mutation ◽  
Transcriptional Target

Abstract RAB GTPases are key players in vesicle trafficking, granule targeting of proteins, granule biogenesis and secretion. RAB31 (Ras-related protein 31) has been implicated in the regulation of vesicular trafficking between the Golgi/TGN and endosomes, and from early endosome (EE) to late endosome (LE). Studies in neuronal cells have implicated RAB31 in the transport of epidermal growth factor receptor (EGFR) from EE to LE. RUNX1, a major hematopoietic transcription factor, plays a critical role in megakaryocyte (MK) maturation and platelet production. Patients with RUNX1 haplodeficiency have thrombocytopenia, abnormal platelet function, and impaired granule contents and secretion. Our studies in a patient with a heterozygous RUNX1 mutation (c.969-323G>T ) (Sun et al, Blood 103: 948-54, 2004) associated with thrombocytopenia, platelet dysfunction, granule deficiency and impaired platelet responses revealed decreased platelet expression of RAB31. The role of RAB31 in MK and platelets is unknown. We addressed the hypothesis that RAB31 is a transcriptional target of RUNX1. We studied the role of RAB31 on vesicle transport in MK cells in human erythroleukemia (HEL) cells transformed into megakaryocytic cells by PMA (phorbol 12-myristate 13-acetate). RAB31 mRNA was decreased on platelet expression profiling of the patient (fold change: 0.28, p<0.0076, Sun et al J Thromb Haemost 5: 146-154). With real-time PCR platelet platelet RAB31 mRNA was decreased compared to 5 healthy controls by 60-80% in our patient (P1) and in two additional unrelated patients (siblings, P2 and P3) with RUNX1 mutation (c.508+1G>A). Platelet RAB31 protein was decreased compared to that in 5 healthy controls in patients P2 and P3. RAB31 promoter region (-2023/-1bp from the ATG) revealed 4 RUNX1 consensus sites: site I (-813/-808), site II (-972/-967), site III (-1500/-1495) and site IV (-2007/-2002). Chromatin immunoprecipitation (ChIP) revealed RUNX1 binding to RAB31 sites II and IV but not to other sites. Electrophoretic mobility shift assays using HEL cell proteins showed RUNX1 binding to sites II and IV. In luciferase reporter assays, mutation of individual sites II and IV decreased promoter activity indicating that they are functional sites. RAB31 promoter activity and protein expression were inhibited by RUNX1 siRNA and enhanced by RUNX1 overexpression. These indicate that RAB31 is a direct RUNX1 target, providing a mechanism for decreased RAB31 in patient platelets. We investigated putative roles of RUNX1 and its target RAB31 in endosomal dynamics in PMA treated HEL cells. We used immunofluorescent staining for markers of early endosomes (EE, EEA1) and late endosomes/multivesicular bodies (LE/MVB, CD63) in RUNX1- or RAB31-depleted cells by siRNA transfection. Either RUNX1 or RAB31 siRNA yielded a striking enlargement of early endosomes, as indicated by the EE marker EEA1. This finding suggests a role for RUNX1/RAB31 in EE maturation, either by mediating vesicle fission, or maturation to late endosomes by fusion with other endosomal vesicles. This effect of RUNX1 knockdown on EE enlargement was partially reversed by reconstitution of RAB31 by plasmid co-transfection, indicating that RAB31 is a significant but non-exclusive contributor to this RUNX1 function. Conclusions: These studies provide the first evidence that RAB31 is a direct transcriptional target of RUNX1 and a mechanism for RAB31 downregulation in RUNX1 haplodeficient patients. Downregulation of RAB31 or RUNX1 results in impaired endosomal maturation/trafficking, and this may contribute to the defective handling of α-granule proteins recognized in patients with RUNX1 mutations. Disclosures Lambert: Sysmex: Consultancy; Rigel: Consultancy; Bayer: Membership on an entity's Board of Directors or advisory committees; Educational Concepts in Medicine: Consultancy; CSL: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Summus: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Shionogi: Consultancy.

Targeting Sp1 Transactivation In Waldenstrom's Macroglobulinemia: a Novel Therapeutic Option

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 120-120
Author(s):  
Mariateresa Fulciniti ◽  
Samir B. Amin ◽  
Varuna Mohan ◽  
Guang Yang ◽  
Puru Nanjappa ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Growth ◽  
Board Of Directors ◽  
Research Funding ◽  
Luciferase Reporter ◽  
Parp Cleavage ◽  
Synergistic Activity ◽  
Advisory Committees ◽  
Growth And Survival ◽  
Cell Growth And Survival

Abstract Abstract 120 The transcription factor Sp1 transactivates expression of genes containing proximal GC/GT-rich promoter elements controlling cell differentiation, cell cycle and apoptosis affecting growth and survival of tumor cells. Based on previous observation that key multiple myeloma (MM) cell growth and survival genes such as NF-kB p65, IGF-IR, VEGF, and IL-6 are controlled by Sp proteins, we have previously investigated and observed high Sp1 expression and activity in MM cells and confirmed its role in MM by Sp1 knock down using both siRNA and lentiviral shRNA constructs specific for Sp1. We further evaluated the role of Sp-1 in WM and observed high nuclear Sp1 protein expression along with increased Sp1 activity in WM cells compared to normal peripheral blood mononuclear cells (PBMC). Moreover, adhesion of WM cells to bone marrow stromal cells (BMSC) further induces Sp1 activity in WM cells. Based on these data, we have investigated the anti-WM activity of Terameprocol (TMP), a small molecule suitable for clinical application,which specifically competes with Sp1-specific DNA binding domains within gene promoter regions. It disrupts the interaction between Sp1 and GC-rich motifs inhibiting Sp1 activity without direct effect on its expression. We have confirmed inhibition of both basal and BMSC-induced binding and transcriptional activity of Sp1 in WM cells using an ELISA assay specific for measuring Sp1 binding activity and using Sp1 sensitive luciferase reporter plasmid. TMP treatment did not affect Sp1 protein levels. Importantly, TMP significantly inhibited WM cell growth in a dose-dependent fashion (IC50 between 5–20 μ M at 24 hours) and was able to overcome the protective effects of BMSCs. TMP activates the mitochondrial apoptotic pathway via induction of caspase-3, -9 and -7 and PARP cleavage but without caspase-8 activation. TMP treatment also led to downregulation of expression of survivin, a known anti-apoptotic gene transcriptionally regulated by Sp1. We have also confirmed in vivo activity of TMP in a murine xenograft model of MM. Finally based on the data suggesting that both dexamethasone and revlimid increase Sp1 activity, we have combined TMP with these agents and observed synergistic activity on cell growth and survival. In conclusion, our results demonstrate Sp1 as an important transcription factor in WM and provides preclinical rationale for clinical development of TMP as a specific Sp1 inhibitor alone and in combination with conventional and novel agents in WM. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Treon:Millennium Pharmaceuticals, Genentech BiOncology, Biogen IDEC, Celgene, Novartis, Cephalon: Consultancy, Honoraria, Research Funding; Celgene Corporation: Research Funding; Novartis Corporation: Research Funding; Genentech: Consultancy, Research Funding. Munshi:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.

Promoter-Wide Transcriptional Deregulation In Waldenstrom Macroglobulinemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3620-3620
Author(s):  
Yang Liu ◽  
Min Ni ◽  
Aldo M. Roccaro ◽  
Xavier Leleu ◽  
Yong Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Binding Sites ◽  
Research Funding ◽  
Enrichment Analysis ◽  
Promoter Regions ◽  
Advisory Committees ◽  
Pol Ii ◽  
Normal Controls

Abstract Abstract 3620 Introduction: Waldenstrom macroglobulinemia (WM) is a rare indolent non-Hodgkin lymphoma, characterized by bone marrow infiltration of clonal lymphoplasmacytic cells. Despite recent advances in understanding the pathogenesis of this disease, the molecular basis of WM etiology has not been clearly defined. We therefore performed genome-wide analysis of RNA polymerase II (pol II) binding sites and gene expression profiling in primary WM cells in order to comprehensively define the aberrant transcriptional regulation and related genes in WM. Methods: Primary CD19+ bone marrow derived WM cells and normal primary bone marrow were used. Genomic DNA was extracted using genome isolation kit (QIAGEN) after cross linking. All the DNA samples were sent for Chip assay and human promoter 1.0R array (Genepathway Inc.) which comprised of over 4.6 million probes tiled through over 25.500 human promoter regions. Each promoter region covers approximately 7.6kb upstream through 2.45kb downstream of the transcription start sites. For over 1,300 cancer associated genes, coverage of promoter regions was expanded to additional genomic content; for selected genes total coverage spans from 10kb upstream through 2.45kb downstream of transcription start sites. The published gene expression datasets (GDS2643) which included 10 CD19+ B cell from bone marrow of 10 WM patients and 8 normal controls was analyzed by d-chip software and normalized to normal control. The motif analysis was performed using Cistrome online tools from the Dana Farber Cancer Institute. The gene sets enrichment analysis (GSEA) was performed using GSEA online software from Broad institute. Results: A total of 13,546 high-confidence pol II sites were identified in WM samples and share a small percentage of overlap (11.5%) with the binding sites identified in normal controls. Combining the expression microarray data of WM patient samples and normal controls, we demonstrated a significant correlation between high levels of gene expression and enriched promoter binding of pol II. Notably, we also observed that the WM-unique pol II binding sites are localized in the promoters of 5,556 genes which are involved in important signaling pathways, such as Jak/STAT and MAPK pathways by applying gene set enrichment analysis (GSEA). Interestingly, we found that STAT, FOXO and IRF family binding sites motifs were enriched in the pol II-bound promoter region of IL-6 which plays a crucial role in cell proliferation and survival of WM cells. Moreover, the CpG island associated c-fos promoter was enriched for Pol II binding as compared to the normal control. Conclusion: The presence of increased Pol II binding and the identification of transcription factor motifs in the promoters of key oncogenes may lead to a better understanding of WM. Our findings suggest that altered transcriptional regulation may play an important role in the pathogenesis of WM. In addition, this study will provide novel insights into the molecular mechanism of WM etiology, and may lead to discovery of novel diagnostic molecular biomarkers and therapeutic targets for WM. Disclosures: Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Novel BET Protein Degrader-Based Combinations Exert Lethality Against Human AML Resistant to BET Inhibitors Due to Increased Activity of β-Catenin-TCF7L2- MYC Axis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 177-177
Author(s):  
Dyana T. Saenz ◽  
Warren Fiskus ◽  
Taghi Manshouri ◽  
David N Saenz ◽  
Raffaella Soldi ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Progenitor Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Genetic Alterations ◽  
Advisory Committees ◽  
Lethal Effects ◽  
Bet Inhibitors ◽  
Nuclear Levels ◽  
And Control

Abstract Bromodomain and extra-terminal protein (BETP) inhibitors (BETis) disrupt the chromatin binding and activity of the BETP BRD4 in facilitating RNA pol II-mediated mRNA transcription, thereby depleting levels of active oncoproteins including c-Myc, CDK6, BCL2, PIM1 and MCL1. BETi treatment also increases protein levels of p21, p27 and HEXIM1, thereby causing growth inhibition and apoptosis of AML blast progenitor cells (BPCs), including post-MPN, secondary AML (sAML) BPCs. Treatment with BETi (e.g., OTX015) has been shown to reduce AML burden and induce clinical remissions. However, BETi-refractory AML develops uniformly. Previous reports utilizing mouse AML models have highlighted that persister-resistance to BETi (BETi-P/R) in AML stem progenitor cells is observed despite BETi treatment and reduction of BRD4 occupancy on the chromatin. This is mediated by re-expression of c-Myc due to transcriptional activity of WNT-β-catenin. In the present studies, we developed human sAML models of BETi-P/R to elucidate the mechanisms and develop targeted therapies against BETi-P/R sAML BPCs. Utilizing human sAML control (parental) SET2 and HEL92.1.7 cells and subjecting them to at least 10 exposures to 1.0 µM of the BETi OTX015 for 48 hours followed by full recovery, we first generated the BETi-P/R SET2-P/R and HEL-P/R cells. These cells were > 10-fold resistant to OTX015 and exhibited cross-resistance to other BETis, including JQ1 and ABBV-075. As compared to the control sAML cells, SET2-P/R and HEL-P/R cells neither exhibited additional genetic alterations by NextGen whole-exome sequencing, nor showed altered levels of TRIM33, SPOP or phosphorylated BRD4 (previously described mechanisms of BETi-resistance). In contrast, compared to the control, SET2-P/R and HEL-P/R cells demonstrated significantly higher nuclear levels and binding of β-catenin to the transcription factor TCF7L2 (TCF4) and TBL1X (TBL1), associated with increased expression of TCF4 targets, including c-Myc, Cyclin D1, TERT and Survivin. ATAC-Seq and ChIP-Seq (H3K27Ac mark) analyses showed significant gain of peaks and active enhancers in HEL-P/R over HEL92.1.7 cells, including enrichment of the STAT5, MYC, PU.1, GATA2 and MYB transcription factor binding sites, as well as newly gained peaks in the enhancers of JAK1/2, RUNX1, PU.1, MYC, BCL2L1 and CTNNB1. RNA-Seq analysis showed significant increase/decrease in mRNA expressions (340/247), with increased expression of gene-sets involving MYC/MAX, STAT5, NFkB and TCF4 targets. QPCR and Western analyses confirmed significant perturbation in gene expressions, with increase in TCF4, c-Myc, Survivin and PIM1 in HEL-P/R over HEL92.1.7 cells. Consistent with the finding that shRNA-mediated knockdown of BRD4 exerted similar lethal effects in BETi-P/R versus control cells, we also discovered that BETP-PROTAC (proteolysis targeting chimera) ARV-771 (Arvinas, Inc.) that degraded BRD4/3/2 was equipotent in inducing apoptosis of BETi-P/R and control sAML cells. Also, consistent with increased nuclear levels and binding (utilizing confocal microscopy) of β-catenin with TBL1 and TCF4 in BETi-P/R sAML BPCs, β-catenin inhibitor BC2059 (Beta-Cat Pharma), which disrupts the binding of nuclear β-catenin with TBL1 and TCF4 and depletes β-catenin levels, exerted similar lethal effects in BETi-P/R sAML and control sAML cells. Consistent with these findings, we also determined that co-treatment with ARV-771 and BC2059 exerted synergistic in vitro lethality against BETi-P/R sAML BPCs (combination indices < 1.0), which was associated with greater reduction in levels of c-Myc, TCF4, Survivin, CDK6, PIM1 and Bcl-xL. Co-treatment with ARV-771 and BC2059 was also synergistically lethal against 12 patient-derived samples of CD34+ sAML BPCs. Notably, compared to treatment with each agent alone or vehicle control, in vivo treatment with ARV-771 (30 mg/kg SQ daily x 5, per week) and BC2059 (30 mg/kg IP BIW per week) for 3 weeks, significantly reduced the sAML burden and improved survival of the NSG mice engrafted with luciferase-transduced HEL-P/R cells (p < 0.01). These findings demonstrate that increased levels and activity of β-catenin-TCF7L2-MYC axis is mechanistically responsible for BETi-P/R, and co-targeting with BETP degrader and β-catenin-TCF4 inhibitor is synergistically lethal against BETi-P/R sAML BPCs. Disclosures Soldi: Beta Cat Pharma: Employment. Bose:Astellas Pharmaceuticals: Research Funding; Celgene Corporation: Honoraria, Research Funding; Blueprint Medicines Corporation: Research Funding; Pfizer, Inc.: Research Funding; Constellation Pharmaceuticals: Research Funding; CTI BioPharma: Research Funding; Incyte Corporation: Honoraria, Research Funding. Kadia:BMS: Research Funding; Takeda: Consultancy; Novartis: Consultancy; Celgene: Research Funding; BMS: Research Funding; Jazz: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy; Celgene: Research Funding; Jazz: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy; Pfizer: Consultancy, Research Funding; Abbvie: Consultancy; Abbvie: Consultancy. DiNardo:Abbvie: Honoraria; Medimmune: Honoraria; Karyopharm: Honoraria; Celgene: Honoraria; Bayer: Honoraria; Agios: Consultancy. Horrigan:Beta Cat Pharma: Employment. Khoury:Stemline Therapeutics: Research Funding. Verstovsek:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy.

scholarly journals Activating KRAS, NRAS, and BRAF Mutants Enhance Proteasome Capacity and Reduce Endoplasmic Reticulum Stress in Multiple Myeloma, Thereby Promoting Plasma Cell Survival and Proteasome Inhibitor Resistance

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 406-406
Author(s):  
Fazal Shirazi ◽  
Richard J. Jones ◽  
Isere Kuiatse ◽  
Zuzana Berkova ◽  
Hua Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Transcription Factor ◽  
Board Of Directors ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Research Funding ◽  
Mek Inhibitor ◽  
Mapk Signaling ◽  
Dominant Negative ◽  
Advisory Committees

Abstract Introduction: Multiple myeloma, a malignant proliferation of differentiated plasma cells, is the second most commonly diagnosed hematologic malignancy, and the number of cases may grow by almost 60% between 2010 and 2030. Recent therapeutic advances, including the use of proteasome inhibitors (PIs), have contributed to a doubling of the median overall survival in myeloma patients. This has been paralleled by an increased understanding of the mutational spectrum in this disease, which was first noted almost three decades ago to harbor KRAS and NRAS mutations. KRAS, NRAS, and BRAF mutations which induce p44/42 Mitogen-activated protein kinase (MAPK) signaling are found in about half of myeloma patients, and seem to contribute to proteasome inhibitor (PI) resistance, but the underlying mechanisms still remains elusive. Methods: ANBL-6 and U266 human-derived myeloma cell lines have endogenous wild-type (WT) KRAS, NRAS, and BRAF, and were used in this study. All cell lines were validated through The MD Anderson Cancer Center Characterized Cell Line Core Facility. We established lines stably expressing WT, constitutively active (CA)(G12V/G13D/Q61H), or dominant negative (DN)(S17N) KRAS and NRAS mutants, or V600E or DN BRAF. Cell viability was evaluated using the WST-1 tetrazolium reagent, while the chymotrypsin-, trypsin- and caspase-like activities were determined using fluorogenic substrates. Results: CA KRAS, NRAS, and BRAF mutants reduced the sensitivity of ANBL-6 and U266 cells to bortezomib and carfilzomib, while their DN variants sensitized cells to both PIs. This was associated with an induction by these CA mutants of the proteasome chymotrypsin-, trypsin- and caspase-like activities, while the DN variants reduced proteasome activity. These activity changes occurred in parallel with increased expression at both the mRNA and protein levels of catalytically active Proteasome subunit beta (PSMB)-8, PSMB9, and PSMB10, and of the proteasome assembly chaperone Proteasome maturation protein (POMP). Mechanistic studies showed that MAPK induction by the CA mutants caused activation of the ETS transcription factor (ELK1), which was found to have consensus binding sites in the promoters of PSMB8, PSMB9, PSMB10, and POMP. Notably, ELK1 suppression reduced PSMB8, PSMB9, PSMB10, and POMP expression, directly linking RAS/RAF/MAPK signaling to proteasome biology, and this suppression enhanced PI sensitivity. Inhibition of MAPK signaling with either the MAPK kinase (MEK) inhibitor selumetinib or the pan-RAF inhibitor TAK-632 showed synergistic activity with either bortezomib or carfilzomib that was more consistent in cell lines harboring CA mutants as opposed to the DN or WT constructs. Combination regimens of selumetinib or TAK-632 with either bortezomib or carfilzomib induced greater inhibition of the proteasome chymotrypsin-, trypsin- and caspase-like activities than the PIs as single agents. Finally, CA KRAS, NRAS, and BRAF mutants reduced expression levels of genes and proteins involved in the unfolded protein response (UPR), including Activating transcription factor (ATF)-4, -5, and -6, as well as C/EBP homologous protein transcription factor (CHOP) and the spliced variant of X-box binding protein 1 (XBP1s). In contrast, their dominant negative counterparts enhanced expression of the UPR effectors, consistent with an increase in endoplasmic reticulum (ER) stress. Conclusion: Taken together, the data support the hypothesis that activating MAPK pathway mutations enhance PI resistance by increasing proteasome capacity, and provide a rationale for targeting such patients with PI/RAF or PI/MEK inhibitor combinations. Moreover, they argue that these mutations promote plasma cell survival by reducing cellular stress, thereby distancing myeloma cells from the apoptotic threshold, potentially explaining their high frequency in myeloma. Disclosures Lee: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies Corporation: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Chugai Biopharmaceuticals: Consultancy; Takeda Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees. Dick:Takeda Oncology: Employment, Equity Ownership. Chattopadhyay:Takeda Oncology: Employment. Orlowski:Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; BioTheryX, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Poseida: Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals FLT3 and LSD1 Inhibitor Combinations Synergistically Repress Growth of FLT3-Mutant Acute Myeloid Leukemia Via Blockage of MYC Function

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-32
Author(s):  
Daniel J. Coleman ◽  
Brittany M. Smith ◽  
Cody Coblentz ◽  
Rowan L. Callahan ◽  
Jake VanCampen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Board Of Directors ◽  
Transcriptional Activation ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Target Genes ◽  
Publicly Traded ◽  
Private Company ◽  
Advisory Committees ◽  
Flt3 Inhibitor

Internal Tandem Duplication mutations of Fms Related Receptor Tyrosine Kinase 3 (FLT3), known as FLT3-ITD mutations, are associated with poor prognosis in Acute Myeloid Leukemia (AML). The clinical efficacy of inhibiting FLT3 in AML is limited by the rapid development of drug resistance and relapse, underscoring a need for more potent and durable treatment strategies. The early persistence of leukemic blasts during FLT3 inhibition is a key driver of resistance. We find that in combination, inhibitors of Lysine Specific Demethylase 1 (LSD1) potentiate the activity of FLT3 inhibitors, driving synergistic cell death. This novel therapeutic approach has the potential to drive deeper therapeutic responses in FLT3-Mutant AML, delaying or preventing the development of resistance. LSD1 is a dynamic DNA-associated protein that functions as a chromatin modifier and transcription factor. LSD1 removes methylation on both lysine 4 of histone H3 (H3K4), associated with transcriptional activation, and lysine 9 (H3K9), associated with transcriptional repression. Additionally, LSD1 has been reported to function as a transcription factor independent of its catalytic demethylase function. LSD1 inhibition reduces cell proliferation in several cancer types. In AML specifically, inhibition of LSD1 has been reported to activate enhancers associated with genes that promote differentiation. We hypothesized that combining LSD1 inhibition with FLT3 inhibition in FLT3-ITD AML would result in synergistic effects on cell viability through reactivating differentiation pathways and more strongly blocking proliferation. In this study, we aimed to examine the efficacy, transcriptional effects, and changes in chromatin dynamics when combining LSD1 inhibition with FLT3 inhibition in a FLT3-ITD mutant cell line and patient samples. We used matrix combination screening to determine that combining the FLT3 inhibitor Quizartinib with LSD1 inhibitors (GSK-2879552 or ORY-1001) synergistically represses cell viability in the FLT3-ITD mutant MOLM-13 cell line and in multiple primary AML samples. RNA-seq followed by Gene Set Enrichment Analysis revealed that combining LSD1 and FLT3 inhibition synergistically represses target genes of the oncogenic transcription factor MYC. This finding was corroborated through high-throughput genome-wide profiling of histone marks, using the recently developed technique Cleavage Under Targets and Tagmentation (CUT&Tag). Specifically, we discovered several promoter regions in which acetylation of lysine 27 of Histone H3 (H3K27Ac), associated with transcriptional activation, was repressed by combining LSD1 and FLT3 inhibition. The genes associated with these regions were strongly enriched for known MYC target genes. Through additional genomic profiling methods including ChIP-seq and ATAC-seq, we have established potential roles for several DNA-binding transcription factors including CEBPA, RUNX1, STAT5, and LSD1 itself, that may mediate repression of MYC function resulting from combining LSD1 and FLT3 inhibition. Together, our work establishes LSD1 and FLT3 inhibitor combinations as a promising treatment strategy in FLT3-ITD AML. Importantly, this study identifies combined FLT3 and LSD1 inhibition as an effective strategy to indirectly target MYC function, as MYC is often referred to as an "undruggable" target. Furthermore, it has the potential to drive deeper molecular responses in FLT3-mutant AML, decreasing the likelihood of treatment resistance. Disclosures Druker: Bristol-Myers Squibb: Research Funding; Blueprint Medicines: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Millipore (formerly Upstate Biotechnology): Patents & Royalties; Pfizer: Research Funding; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Patient True Talks: Consultancy; Oregon Health & Science University: Patents & Royalties; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; MolecularMD (acquired by ICON): Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Iterion Therapeutics (formerly Beta Cat Pharmaceuticals): Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics Inc. (formerly Lorus): Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; GRAIL: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; Leukemia & Lymphoma Society: Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Dana-Farber Cancer Institute: Patents & Royalties; EnLiven: Consultancy, Research Funding. Maxson:Gilead Sciences: Research Funding; Ionis Pharmaceuticals: Other: Joint oversight committee for a collaboration between OHSU and Ionis Pharmaceuticals.

scholarly journals E2F1 Is a Biomarker of Selinexor Resistance in Relapsed/Refractory Multiple Myeloma Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3216-3216 ◽  
Author(s):  
Alessandro Lagana ◽  
Seongjee Park ◽  
Donna Edwards ◽  
Violetta Leshchenko ◽  
Marsha Crochiere ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Transcription Factor ◽  
Board Of Directors ◽  
Nuclear Export ◽  
Research Funding ◽  
Differential Expression Analysis ◽  
Negative Regulator ◽  
Rapid Progression ◽  
Advisory Committees ◽  
Nuclear Retention

Abstract Selinexor (KPT-330) is a selective inhibitor of nuclear export (SINE) which specifically targets XPO1 (Exportin 1)-mediated nuclear export, leading to increased nuclear retention of major tumor suppressor proteins and inducing selective apoptosis in cancer cells. Several phase I and II clinical trials demonstrate evidence of anti-cancer activity of Selinexor in solid tumors (i.e metastatic prostate cancer (PMID: 29487219), advanced refractory bone or soft tissue sarcoma (PMID: 27458288) and non-small cell lung cancer (PMID: 28647672); as well as, hematological malignancies, including non-Hodgkin lymphoma (PMID: 28468797), acute myeloid leukemia (PMID: 29304833) and multiple myeloma (MM) (PMID: 29381435). In the STORM (Selinexor Treatment of Refractory Myeloma) trial, the combination of Selinexor with dexamethasone in MM patients refractory to bortezomib, carfilzomib, lenalidomide and pomalidomide (quad-refractory), or in addition, to daratumumab (penta-refractory), has shown an overall response rate (ORR) of 21% (Vogl et al, JCO 2018). Our objective is to identify biomarkers for selection of patients at higher likelihood of clinical benefit from Selinexor salvage and understand mechanisms of Selinexor resistance. We therefore analyzed transcriptional differences using RNA sequencing in CD138+ cells from bone marrow aspirates obtained prior to treatment from 32 MM patients enrolled in STORM. The raw data (fastq) was mapped by using the tool STAR and gene-level annotated by featureCounts. Patients were split in two groups based on their progression-free survival (PFS). Differential expression analysis was performed using the tool DESeq2, which enables a more quantitative analysis of comparative RNA-seq data using shrinkage estimators for dispersion and fold change. The results revealed significant up-regulation of 13 genes in patients with PFS < 120 days (n = 21, p < 0.05) versus patients with PFS > 120 days (n=11), including the transcription factor E2F1 and its targets MYBL2, FANCA, GINS3 and SLX4 (Fig. 1). Next, we evaluated the expression of E2F1 in another set of 26 patients from the STORM trial by Affymetrix U133 gene expression microarrays. Data was analyzed using the Signal Space Transformation (SST)-Robust Multi-Chip Analysis (RMA) algorithm. Patients with PFS < 120 days (n = 19) exhibited significant up-regulation of E2F1 (p < 0.05) (Fig. 2). E2F1 is a transcription factor that regulates cell cycle G1/S progression. At rest, E2F1 is complexed with its negative regulator retinoblastoma(RB) protein. Upon phosphorylation of RB by the Cyclin D1-CDK4/6 complex, pRB is inactivated allowing E2F1 to commence transcription of target genes allowing G1/S progression. E2F transcription factors are exported by XPO1 from the nucleus to the cytoplasm. We treated RPMI8226 (IC50=150nM) and MM1S (IC50=25nM) human myeloma cell lines with Selinexor at IC50 and examined nuclear vs cytoplasmic expression of E2F1 after 24 and 48 hours by western blotting. Our results demonstrated nuclear retention of E2F1 following treatment of HMCLs with Selinexor and suggest a model where overexpression of E2F1 overwhelms the nuclear export mechanism and may result in downstream gene programming that confers a proliferative advantage in cells, manifested by rapid progression (<120 days) in patients. Our findings suggest a model where E2F1 expression may be a biomarker of Selinexor resistance. We are currently validating our findings in additional samples from patients with MM treated with Selinexor. Disclosures Crochiere: Karyopharm Therapeutics Inc: Employment. Landesman:Karyopharm Therapeutics Inc: Employment. Chari:Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Array Biopharma: Research Funding; Bristol Myers Squibb: Consultancy; Pharmacyclics: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; The Binding Site: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Cho:Janssen: Consultancy; Genentech Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; J & J: Consultancy; Agenus Inc.: Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees. Barlogie:Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; Millenium: Consultancy, Research Funding; Dana Farber Cancer Institute: Other: travel stipend; International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; Celgene: Consultancy, Research Funding; Multiple Myeloma Research Foundation: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend. Jagannath:Multiple Myeloma Research Foundation: Speakers Bureau; Merck: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Medicom: Speakers Bureau.

scholarly journals MCL-1 and PKA/AMPK Axis Fuel Venetoclax Resistance in Lymphoid Cancers

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1284-1284
Author(s):  
Vivian M. Liu ◽  
Romain Guièze ◽  
Daniel Rosebrock ◽  
Alexis A Jourdain ◽  
María Hernández-Sánchez ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Safety Monitoring ◽  
Data Safety Monitoring Board ◽  
Advisory Board ◽  
Advisory Committees ◽  
Data Safety ◽  
Genome Wide

Venetoclax, the first approved BH3 mimetic targeting BCL2, demonstrates high response rate in chronic lymphocytic leukemia (CLL) but resistant cases are emerging. Aside from BCL2 mutations affecting venetoclax binding, multiple lines of mounting evidence suggest a role for non-mutational mechanisms underlying resistance to this drug. By applying both CRISPR-Cas9 knock-out and ORF overexpression screens in the lymphoma cell line OCI-Ly1, we previously reported the identification of MCL-1 overexpression and of the AMPK/PKA signaling axis in altering energy metabolism underlying venetoclax resistance (Guieze, ASH 2018). Here, we report further in-depth exploration of the impact of these findings, discovered through the analysis of lymphoid cell lines, and of specimens collected from CLL patients developing venetoclax resistance. The resistant lymphoma cell lines that we generated (OCI-Ly1 and SU-DHL4 cells) displayed increased oxidative phosphorylation (OXPHOS) compared to the parental lines, measured by Seahorse assay. We instead observed that venetoclax rapidly perturbs OXPHOS in sensitive cells. This process is dependent on mitochondrial outer membrane permeabilization, as this effect is abrogated in BAX/BAK1 double knockout (KO) cells. Targeting OXPHOS was shown to synergize with venetoclax in vitro and in vivo, as combination of venetoclax and oligomicin (an inhibitor of the ATP synthase, the complex V of the mitochondrial electron transport chain), was more effective than each drug alone in reducing tumor growth of a subcutaneous NSG xenograft model based on OCI-Ly1. Among the candidate markers driving resistance identified from the genome-wide screens, we focused on AMP pathway members (AMPK and PKA) and the ID3 transcriptional regulator, given that ID3 KO cells demonstrated similar transcriptomic changes than the resistant OCI-Ly1 cells. We found that PRKAR2B (encoding a PKA subunit), already highlighted in our ORF screen, was the top transcript overexpressed when knocking out ID3. To clarify how the dominant-negative transcription factor ID3 regulates PRKAR2B expression, we performed ATAC-seq of the ID3 OCI-Ly1 knockout (vs control) lines in order to determine differential signatures of chromatin accessibility and transcription factor engagement. We showed that ID3 repression leads to genome-wide increased accessibility associated with motifs of the lymphoid transcription factor TCF3. TCF3 has previously been shown to interact with ID3 and to be involved in the transcription of ADIPOQ, which was identified in the GOF screen. TCF3 binding sites were confirmed to be present within putative enhancer regions of PRKAR2B in a B cell context. We then investigated whether our findings could be validated in patient samples. By whole-exome sequencing of matched pretreatment and venetoclax-resistant CLL samples collected from 6 patients, we did not detect any recurrent somatic mutations associated with resistance. The resistant samples from three of 6 patients, however, harbored subclones with 1q amplification in a common region encompassing the MCL1 locus. We identified 4 additional CLL cases relapsing on venetoclax with leukemia samples collected before and after relapse. By immunohistochemical staining of 9 of 10 cases for which tissue was available, we detected increased MCL-1 expression at relapse in 6 of 9 cases (p = 0.026). We furthermore confirmed the involvement of AMPK signaling by detecting evidence of AMPK, ACC and p-ACC expression in 4 of 9 patients (all p = 0.0062). ID3 expression was decreased at matched relapse samples (p = 0.0001), supporting the presence of the resistance circuit we identified above. Taken together, our results identified the increased MCL-1 expression and PKA/AMPK activation as underlying mechanisms for venetoclax resistance. Our data support the implementation of combinatorial therapy with metabolic modulators to address venetoclax resistance. Disclosures Guièze: Abbvie: Honoraria; Roche: Honoraria; Janssen: Honoraria; Gilead: Honoraria. Thompson:AbbVie: Research Funding; Amgen: Consultancy, Research Funding; Pfizer: Research Funding; Pharmacyclics: Research Funding; Genentech: Consultancy, Honoraria; Gilead: Consultancy, Honoraria. Davids:AbbVie, Acerta Pharma, Adaptive, Biotechnologies, Astra-Zeneca, Genentech, Gilead Sciences, Janssen, Pharmacyclics, TG therapeutics: Membership on an entity's Board of Directors or advisory committees; Research to Practice: Honoraria; AbbVie, Astra-Zeneca, Genentech, Janssen, MEI, Pharmacyclics, Syros Pharmaceuticals, Verastem: Consultancy; Acerta Pharma, Ascentage Pharma, Genentech, MEI pharma, Pharmacyclics, Surface Oncology, TG Therapeutics, Verastem: Research Funding. Brown:AbbVie: Consultancy; Acerta Pharma: Consultancy; Loxo: Consultancy, Research Funding; BeiGene: Consultancy; Catapult Therapeutics: Consultancy; AstraZeneca: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Pharmacyclics: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy, Research Funding; Sun Pharmaceuticals: Research Funding; Janssen: Honoraria; Teva: Honoraria; Morphosys: Other: Data safety monitoring board; Invectys: Other: Data safety monitoring board; Octapharma: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding; Juno/Celgene: Consultancy; Dynamo Therapeutics: Consultancy; Genentech/Roche: Consultancy; Gilead: Consultancy, Research Funding. Wierda:Xencor: Research Funding; Cyclcel: Research Funding; Genentech: Research Funding; Pharmacyclics LLC: Research Funding; Gilead Sciences: Research Funding; KITE pharma: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Sunesis: Research Funding; AbbVie: Research Funding; Janssen: Research Funding; Acerta Pharma Inc: Research Funding; GSK/Novartis: Research Funding; Miragen: Research Funding; Loxo Oncology Inc.: Research Funding; Juno Therapeutics: Research Funding. Letai:AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding; Zeno Pharmaceuticals, Vivid Bioscience, Flash Therapeutics, Dialectic Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Cofounder or Advisory Board member. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Mootha:Jansen Pharmaceuticals: Other: SAB, compensation; 5am Ventures: Other: SAB, compensation; Raze Therapeutics: Other: Founder, SAB, equity. Getz:MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents & Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER; Pharmacyclics: Research Funding; IBM: Research Funding. Wu:Pharmacyclics: Research Funding; Neon Therapeutics: Other: Member, Advisory Board.

scholarly journals Type 1 Calreticulin Mutations Differentially Activate the IRE1α-XBP1 Pathway of the Unfolded Protein Response to Drive Myeloproliferative Neoplasms

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 628-628
Author(s):  
Juan Ibarra ◽  
Yassmin Elbanna ◽  
Katarzyna Kurylowicz ◽  
Harrison S Greenbaum ◽  
Maria Evers ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Binding Sites ◽  
Calcium Binding ◽  
Research Funding ◽  
Advisory Committees ◽  
Calcium Binding Sites ◽  
Er Calcium ◽  
Support Research

Abstract Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR). 80% of CALR mutations are classified as either type 1 or type 2, exemplified by a 52 bp deletion (CALRdel52) and a 5 bp insertion (CALRins5), respectively. Despite their shared mutant C-termini and mutual ability to bind and activate MPL, patients with type 1 and type 2 CALR mutations display significant clinical and prognostic differences. Type 1 mutations are primarily associated with an MF phenotype and a higher risk of fibrotic transformation from ET, while type 2 mutations are more common in ET. Molecularly, type 2 CALR mutant proteins retain many of the calcium binding sites present in the wild type protein, while type 1 CALR mutant proteins lose these residues. The functional consequences of this differential loss of calcium binding sites remain yet unexplored. Current targeted therapies for CALR mutated MPN are not curative, and treatment does not differentiate between type 1 versus type 2 mutant CALR-driven disease, despite the different phenotypic and prognostic outcomes in these patients. In order to improve treatment strategies for CALR mutated MPN patients, it is critical to identify specific dependencies unique to each CALR mutation type that can be exploited for therapeutic gain. Here, we show that type 1 CALRdel52 but not type 2 CALRins5 mutations lead to activation of and dependency on the IRE1α-XBP1 pathway of the unfolded protein response (UPR). Mechanistically, we found that the loss of calcium binding residues in the type 1 mutant CALR protein directly impairs its calcium binding ability, which in turn leads to depleted ER calcium and subsequent activation of the IRE1α-XBP1 pathway. Using cell lines and primary MPN patient samples, we identified two novel transcriptional targets of XBP1 specific to type 1 CALRdel52-expressing cells - the anti-apoptotic protein BCL-2 and the calcium efflux channel IP3R. We show that BCL-2 acts downstream of XBP1 to promote survival in the face of depleted ER calcium, while IP3R is up-regulated downstream of XBP1 to promote continued ER calcium efflux in order to sustain IRE1α-XBP1 pathway activation and survival. We found that genetic or pharmacological inhibition of IRE1α-XBP1 signaling induced cell death only in type 1 mutant but not type 2 mutant or wild type CALR-expressing cells. Moreover, we show that in vivo inhibition of IRE1α significantly abrogates type 1 mutant CALR-driven disease in a bone marrow transplantation model, but has no effect on type 2 mutant CALR-driven disease. This work is the first to demonstrate that type 1 and type 2 mutant CALR-expressing cells display differential molecular dependencies that can be exploited for therapeutic gain. Moreover, this study answers an enduring question regarding the functional consequence of the loss of calcium binding sites on the type 1 mutant CALR protein, and demonstrates how type 1 CALR mutant-expressing cells rewire the UPR, downstream calcium signaling, and apoptotic pathways to drive MPN. Figure 1 Figure 1. Disclosures Koschmieder: BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Shire: Honoraria, Other; Karthos: Other: Travel support; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Geron: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Abbvie: Other: Travel support; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Alexion: Other: Travel support; Sanofi: Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Baxalta: Membership on an entity's Board of Directors or advisory committees, Other; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); CTI: Membership on an entity's Board of Directors or advisory committees, Other; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; AOP Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Image Biosciences: Other: Travel support.

scholarly journals Constitutively Activated STAT3 Induces the Expression of Gli1 in Chronic Lymphocytic Leukemia Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3018-3018
Author(s):  
Uri Rozovski ◽  
David M. Harris ◽  
Ping Li ◽  
Zhiming Liu ◽  
Taghi Manshouri ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Binding Sites ◽  
Research Funding ◽  
Gene Promoter ◽  
Janus Kinase ◽  
Mrna Levels ◽  
Advisory Committees ◽  
Gli1 Expression ◽  
Treatment Naïve ◽  
Induced Apoptosis

The transcription factor glioma associated oncogene (Gli)-1 is a downstream effector of the Hedgehog (HH) signaling pathway. The HH pathway plays a critical role in embryonic development, and in maintenance of stem cells, tissue homeostasis, hematopoiesis, and immune response in the adult organism. Constitutive activation of the HH pathway has been implicated in the development of various cancer types. A recent study of blood samples from 841 treatment-naïve CLL patients identified activated Gli1 in 49%, and mutations in HH pathway genes, that induce activation of Gli1, in 11% of the patients (Ghia et al. Blood 1019). However what induces the expression of Gli1 in CLL cells that do not carry a mutation(s) in the HH pathway has not been elucidated. Because inhibition of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT)-3 pathway in liver cells inhibits the transcription of Gli1 (Zhang et al. J Cell Mol Med 2018), and because in CLL cells STAT3 is constitutively phosphorylated on serine 727 residues and activates genes known to be activated by phosphotyrosine STAT3, we postulated that constitutively activated STAT3 induces the expression of Gli1 in CLL cells. To test our hypothesis we first performed western immunoblotting and found high levels of Gli1 in cell lysates from 6 of 7 CLL patients but not in normal B cells, and using flow cytometry we found that 76% of CD5+/CD19+ CLL cells expressed intracellular Gli1 and that intracellular phosphoserine STAT3 and Gli1 are co-expressed in 70.4% of CLL cells. Then, using chromatin immunoprecipitation (ChIP) we found that STAT3 protein co-immunoprecipitated with DNA of Gli1 and of the STAT3-target genes C-Myc, ROR1, VEGF, and STAT3. Using sequence analysis we identified in the Gli1 gene promoter 5 γ-interferon activation sequence (GAS)-like elements, known as putative STAT3-binding sites, spanning within 930 base pairs upstream the Gli1 start codon. To determine which putative binding site binds STAT3 we designed 5 different probes. Using ChIP we found that STAT3 protein co-immunoprecipitated 4 of 5 STAT3-putative binding sites at high affinity. Then, using an electromobility shift assay (EMSA) of CLL cell nuclear extracts from 3 different patients we detected STAT3-Gli1 DNA complexes with DNA probes designed to detect the PTX3 gene promoter STAT3-binding sites and found that anti-STAT3 antibodies significantly attenuated the binding, confirming that STAT3 binds to the Gli1 gene promoter. To further confirm these findings, we transfected CLL cells with STAT3-short hairpin (sh)RNA and found that STAT3-shRNA significantly downregulated STAT3 and Gli1 mRNA levels. Because Gli1 expression was associated with disease progression (Ghia et al. Blood 1019), we wondered whether Gli1 provides CLL cells with survival advantage and whether inhibition of Gli1 would affect the viability of CLL cells. To answer this question we transfected CLL cells from two different treatment-naïve CLL patients with Gli1 small interfering (si)RNA and, using annexin V/PI, found that Gli1-siRNA induced apoptosis of CLL cells. To further confirm these data we incubated CLL cells with GANT61, a small molecule that inhibits Gli1, and, as previously reported, found that GANT61 increases the spontaneous apoptosis rate of CLL cells, suggesting that inhibition of Gli1 might benefit patients with CLL. In conclusion, we found that constitutively phosphorylated STAT3 activates the Gli1 gene promoter and induced the production of Gli1 protein in CLL cells. In addition, we found that downregulation of Gli1 expression or inhibition of Gli1 induced apoptosis of CLL cells, suggesting that Gli1 is a target for therapy of CLL. Disclosures Burger: BeiGene: Research Funding; Janssen Pharmaceuticals: Consultancy, Honoraria; AstraZeneca: Honoraria; Aptose Biosciences, Inc: Research Funding; Pharmacyclics, an AbbVie company: Research Funding; Gilead Sciences: Research Funding. Bose:Blueprint Medicine Corporation: Consultancy, Research Funding; Incyte Corporation: Consultancy, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Research Funding; Kartos: Consultancy, Research Funding; Constellation: Research Funding; Pfizer: Research Funding; Astellas: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; CTI BioPharma: Research Funding. Thompson:Gilead: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Pharmacyclics: Research Funding; Pfizer: Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Research Funding. Jain:Pharmacyclics, an AbbVie company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Precision Biosciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Verstovsek:NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Incyte: Research Funding; Roche: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy; CTI BioPharma Corp: Research Funding; Gilead: Research Funding; Promedior: Research Funding. Wierda:Gilead Sciences: Research Funding; Cyclcel: Research Funding; Sunesis: Research Funding; Acerta Pharma Inc: Research Funding; AbbVie: Research Funding; KITE pharma: Research Funding; Xencor: Research Funding; Janssen: Research Funding; GSK/Novartis: Research Funding; Miragen: Research Funding; Genentech: Research Funding; Juno Therapeutics: Research Funding; Pharmacyclics LLC: Research Funding; Loxo Oncology Inc.: Research Funding; Oncternal Therapeutics Inc.: Research Funding.

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