Small Molecule PRIMA-1 met Sensitizes Waldenstrom Macroglobulinemia Cells To Apoptosis and Displays Synergistic Cytotoxicity With Bortezomib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5143-5143
Author(s):  
Mona Sobhani ◽  
Kim Kwan ◽  
Manujendra N. Saha ◽  
Christine I. Chen ◽  
Hong Chang
Keyword(s):  
Bone Marrow ◽  
Western Blot ◽  
Cell Viability ◽  
Small Molecule ◽  
Mtt Assay ◽  
Research Funding ◽  
Mutant P53 ◽  
Haematological Malignancies ◽  
Wild Type ◽  
Qrt Pcr

Abstract Background Waldenstrom macroglobulinemia (WM) is a lymphoplasmacytic lymphoma characterized by heterogeneous infiltration of bone marrow and IgM monoclonal gammopathy. WM constitutes 1-2% of haematological malignancies and patients exhibit diverse outcome. Given the current therapies WM remains incurable with high rate of relapse, therefore, the quest for finding a more effective drug continues. PRIMA-1met is a small molecule with the ability to restore wild type conformation to mutant P53 and to activate wild type P53. PRIMA-1met has shown significant anti-tumour activity both in vitro and in vivo by inducing apoptosis in several human cancers including haematological malignancies and is currently under phase I/II clinical trials. To date, the effects of PRIMA-1met have not been explored in WM. Here we examined the antitumor activity of PRIMA-1met alone and in combination with dexamethasone or bortezomib in WM. Design WM cell lines, BCWM-1(Wild type P53) and MWCL-1 (mutant P53), were used for these studies. Cellular proliferation, viability, migration and percentage of cells undergoing apoptosis in WM cells treated with PRIMA-1met or DMSO control were evaluated using trypan blue, MTT assay, Boyden chamber assay and Annexin-V staining methods respectively. The status of key role players in apoptosis and cell cycle regulators were examined following treatment with PRIMA-1met using qRT-PCR and Western blot analyses. Cell viability was also investigated by combining PRIMA-1metwith either dexamethasone or bortezomib in WM cells by MTT assay. Result Both BCWM-1 and MWCL-1 cells showed significant decrease in cell viability after exposure to escalating doses of PRIMA-1met with observed IC50 of 30 μM in BCWM-1 cells and 28 μM in MWCL-1 cells after 48 hours. Two primary WM patients’ samples exposed to increasing concentration of PRIMA-1met demonstrated significant decrease in cell viability with IC50 of 30 and 10 μM after 48 hours. Importantly, peripheral and bone marrow mononuclear cells of 3 healthy donors were subjected to PRIMA-1met and no significant cytotoxicity was observed. Both cell lines showed an increase in apoptosis after PRIMA-1met treatment in a dose-dependent manner compared to control DMSO treatment. In BCWM-1, cell proliferation, measured by counting viable cells after 7 days, was significantly inhibited by PRIMA-1met compared to DMSO control (∼54% fewer cells when treated with 25 μM PRIMA-1met, p<0.05). Decrease in cell migration by as much as 28% at 25 μM compared to DMSO control (p<0.001) was also observed after 8 hours of treatment with PRIMA-1met. Expressions of P53 and its downstream target p21 were elevated in PRIMA-1met treated cells compared to DMSO control. In addition, qRT-PCR studies of 50 genes associated with apoptosis also demonstrated enhanced expression of pro-apoptotic markers such as PUMA, Noxa, caspase 8 and 9, BIM and BAD, which were further confirmed for their protein expressions by Western blot analysis. Finally, PRIMA-1met (25 μM) was found to exert synergistic cytotoxic effect in combination with dexamethasone (2 μM) in MWCL-1 cells (Combination index (CI)=0.5). PRIMA-1met (25 μM) also displayed synergism with bortezomib (3 μM) in its cytotoxicity toward BCWM-1 cells (CI=0.8). Conclusions Our results demonstrate that PRIMA-1met induces apoptosis irrespective of P53 status in WM cells. It also provides the preclinical framework for evaluation of PRIMA-1met either alone or in combination with bortezomib or dexamethasone as a novel therapeutic approach for the treatment of WM patients. Disclosures: Chen: Roche: Honoraria; Johnson & Johnson: Consultancy, Research Funding; Lundbeck: Consultancy; Celgene: Consultancy, Research Funding; GlaxoSmithKline: Research Funding.

Requirement of CREB in Normal and Malignant Hematopoiesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1168-1168
Author(s):  
Jerry C. Cheng ◽  
Deepa Shankar ◽  
Stanley F. Nelson ◽  
Kathleen M. Sakamoto
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Western Blot ◽  
Blot Analysis ◽  
Hematopoietic Cells ◽  
Wild Type ◽  
Qrt Pcr ◽  
T315i Mutation

Abstract CREB is a nuclear transcription factor that plays an important role in regulating cellular proliferation, memory, and glucose homeostasis. We previously demonstrated that CREB is overexpressed in bone marrow cells from a subset of patients with acute leukemia at diagnosis. Furthermore, CREB overexpression is associated with an increased risk of relapse and decreased event-free survival in adult AML patients. Transgenic mice that overexpress CREB in myeloid cells developed myeloproliferative/myelodysplastic syndrome after one year. To further understand the role of CREB in leukemogenesis and in normal hematopoiesis, we employed RNA interference methods to inhibit CREB expression. To achieve sustained, CREB-specific gene knockdown in leukemia and normal hematopoietic cells, a lentiviral-based small hairpin (shRNA) approach was taken. Three CREB specific shRNAs were generated and tested for efficiency of gene knockdown in 293T cells. Knockdown efficiency approached 90 percent by Western blot analysis compared to vector alone and luciferase controls. Human myeloid leukemia cell lines, K562, TF1, and MV411, were then infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed using quantitative real time (qRT)-PCR, Western blot analysis, and growth and viability assays. Lentiviral CREB-shRNA achieved between 50 to 90 percent knockdown of CREB compared to control shRNAs at the protein and mRNA levels. To control for non-specific effects, we performed qRT-PCR analysis of the interferon response gene, OAS1, which was not upregulated in cells transduced with CREB shRNA constructs. Within 72 hours, cells transduced with CREB shRNA had decreased proliferation and survival. Similar results were obtained with murine leukemia cells (NFS60 and BA/F3 bcr-abl).To study the role of CREB in normal hematopoiesis, both primary murine and human hematopoietic cells were transduced with our shRNA constructs, and methylcellulose-based colony assays were performed. Primary hematopoietic cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in colony number compared to control virus-infected cells (p&lt;0.05). Bone marrow colonies consisted of myeloid progenitor cells that were mostly Mac-1+ by FACs analysis. Interestingly, there were fewer differentiated cells in the CREB shRNA transduced cells compared to vector control or wild type cells, suggesting that CREB is critical for both myeloid cell proliferation and differentiation. To study the in vivo effects of CREB knockdown on leukemia progression, we studied mice injected with BA/F3 cells that express both bcr/abl with the T315I mutation and a luciferase reporter gene. BA/F3 cells expressing the T315I mutation have a 2-fold increase in CREB overexpression compared to wild-type cells. Disease progression was monitored using bioluminescence imaging with luciferin. CREB knockdown was 90 percent after transduction and prior to injection into SCID mice. We observed improved survival of mice injected with CREB shRNA transduced BA/F3 bcr-abl (T315I) compared to vector control cells. To understand the mechanism of growth suppression resulting from CREB downregulation, we performed microarray analysis with RNA from CREB shRNA transduced K562 and TF1 cells. Several genes were downregulated using a Human Affymetrix chip. Most notable was Beclin1, a tumor suppressor gene often deleted in prostate and breast cancer that has been implicated in autophagy. Our results demonstrate that CREB is required for normal and leukemic cell proliferation both in vitro and in vivo.

MLN4924, an Investigational Small Molecule Inhibitor of NEDD8-Activating Enzyme (NAE), Inhibits NF-κB Activity and Induces G1 Cell Cycle Arrest and Apoptosis in Pre-Clinical Models of Hodgkin Lymphoma (HL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3717-3717
Author(s):  
Matthew J. Barth ◽  
Cory Mavis ◽  
Francisco J. Hernandez-Ilizaliturri ◽  
Myron S. Czuczman
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Western Blot ◽  
Cell Viability ◽  
Tumor Cells ◽  
Cell Lines ◽  
Small Molecule ◽  
Wild Type ◽  
Cycle Arrest ◽  
Molecule Inhibitor

Abstract Abstract 3717 The incorporation of combined-modality therapy, risk-stratified chemotherapy selection, high-dose chemotherapy and autologous stem cell support (HDC-ASCS), and monitoring treatment response by functional imaging are factors that have contributed to the improvement in clinical outcomes in HL patients. Unfortunately, those patients not eligible for or that have failed HDC-ASCS remain a challenge for the treating oncologist, stressing the need for novel therapeutic strategies. Significant improvements in the understanding of the biology of HL have been achieved, including cellular pathways altered in HL (e.g. the ubiquitin-proteasome system) and the role of the tumor microenvironment. MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE). NAE is an enzyme responsible for activating NEDD8, an ubiquitin-like molecule in the neddylation cascade that is responsible for cullin-ring ligase (CRL) mediated polyubiquitination of proteins targeting them for proteasomal degradation. In order to better understand the activity of MLN4924 in HL, we performed pre-clinical testing in IkB wild type (L-1236), IkB mutated (KM-H2 and L-428) HL cell lines, and in primary tumor cells derived from a HL patient. Malignant cells were exposed to escalating doses of MLN4924 and changes in cell viability were quantified at different time periods by alamar Blue reduction assay. Patient tumor cells were incubated with MLN4924 for 48 hrs and cell viability was determined using the CellTiterGlo assay. Induction of apoptosis in HL cell lines following exposure to MLN4924 was determined by flow cytometry for Annexin-V and propidium iodide (PI) staining and western blot for caspase-3 and PARP cleavage. Cell cycle analysis was performed by flow cytometry using PI staining. Inhibition of NAE by MLN4924 in HL cell lines was measured by western blot for NEDD8-cullin. Finally, changes in NF-kB activity following MLN4924 exposure were determined by p65 nuclear localization using Image stream technology. MLN4924 exhibited a dose- and time-dependent decrease in cell viability in all HL cell lines at nM concentrations. No differences in anti-tumor activity were observed between IkB-wild type (L-1236 IC50 = 250nM) and IkB–mutated HL cell lines (KM-H2 IC50 = 250nM and L-428 IC50 = 300nM). MLN4924 induced apoptosis in a dose-dependent manner in all cell lines tested. In addition, MLN4924 induced cell cycle arrest in G1 phase and inhibition of NAE was demonstrated by a decrease in NEDD8 conjugated CRL. L1236 cells exposed to MLN4924 also demonstrated a decrease in degradation of IκBα as evidenced by increased levels of p-IκBα following exposure to MLN4924 with a corresponding decrease in p65 nuclear translocation. Surprisingly KMH-2 cells, which carry a mutated IκBα protein that is truncated and non-functional, had a decrease in nuclear p65 following exposure to MLN4924, suggesting an alternative mechanism of NF-kB inhibitory activity by MLN4924. In summary, MLN4924 demonstrates activity against HL cells in vitro through inhibition of NF-kB, and is a promising novel agent for the treatment of HL. We continue to investigate the pre-clinical activity of MLN4924 both as a single-agent and in combination with traditional chemotherapy and other novel agents. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Non-Coding HOX Fusions in Pediatric Non-Down Syndrome Acute Megakaryoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 533-533
Author(s):  
Fabienne R.S. Adriaanse ◽  
Sadie M. Sakurada ◽  
Shondra M. Pruett-Miller ◽  
Ronald W. Stam ◽  
Michel C Zwaan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Hox Genes ◽  
Loss Of Function ◽  
Wild Type ◽  
Acute Megakaryoblastic Leukemia ◽  
Male And Female ◽  
Megakaryoblastic Leukemia ◽  
Qrt Pcr ◽  
Gender Specific

The homeobox (HOX) genes are a highly conserved family of transcription factors involved in embryonic patterning as well as adult hematopoiesis. Dysregulation of HOX genes, in particular upregulation of HOXA cluster genes, is a frequent event in Acute Myelogenous Leukemia (AML). Recently, we performed a detailed genomic analysis on pediatric non-Down Syndrome Acute Megakaryoblastic Leukemia (non-DS-AMKL) and identified novel fusions involving a HOX cluster gene in 14.9% of the cases. While most fusions were predicted to lead to an in-frame functional protein, several fusions included a non-coding HOX antisense gene (PLEK-HOXA11-AS, C8orf76-HOXA11-AS, HOXA10-AS-CD164) that were predicted to result in a loss of function of these regulatory transcripts. The functional consequence of these events, however, remain unknown. HOXA11-AS (human) and Hoxa11os (mouse) have been previously shown to have mutually exclusive expression with the Hoxa11 transcript throughout development. We therefore hypothesized that loss of function of non-coding HOX antisense genes as a result of these structural variations would cause upregulation of nearby coding HOXA genes that in turn promote leukemogenesis. To test this hypothesis, using CRISPR-Cas9 technology, we genome edited the human AMKL cell line CMK to carry the PLEK-HOXA11-AS translocation. qRT-PCR of HOXA11-AS and HOXA9-11 transcripts in this cell line recapitulated the pattern seen in patient specimens. Specifically, HOXA11-AS expression was significantly diminished while HOXA10 and HOXA11 transcripts were upregulated 1.8-2.5-fold when compared to parental CMK cells (p=0.0385 and p=0.006 respectively). To further investigate the loss of HOXA11-ASin vivo a CRISPR-Cas9 Hoxa11os knockout mouse model was established. qRT-PCR on bone marrow confirmed the loss of Hoxa11os transcripts in heterozygous (Hoxa11os1+/-) and homozygous (Hoxa11os-/-) mice of both genders (p=&lt;0.0001-0.0012). Consistent with Hoxa11os knockdown, Hoxa11 transcripts were upregulated in male (1.8-fold p=0.0023 Hoxa11os+/-, and 2-fold p=0.0052 Hoxa11os-/-)and female (1.3-fold p=0.0074 Hoxa11os+/- and 2.2-fold p=0.0226 Hoxa11os-/-) bone marrow compared to wild type gender matched littermates. Interestingly, flow cytometry analysis of progenitor subsets revealed gender specific findings. We found a significant increase in the frequency of the lineage negative, Sca-1 and c-Kit positive (LSK) population in males (0.13% of total bone marrow Hoxa11os+/+, 0.19% p=0.0214 Hoxa11os+/-, and 0.25% p=0.0001 Hoxa11os-/-) compared to wild type male littermates but not in female mice at 8 weeks of age. In contrast an increase in the megakaryocyte-erythroid (MEP) population was seen only in the female setting (0.07% Hoxa11os+/+, 0.15% p=0.0055 Hoxa11os+/-, and 0.165% p&lt;0.0001 Hoxa11os-/-). Limiting dilution colony forming assay confirmed the higher LSK frequency with a 2-fold increase in the number of colonies for male knockout marrow compared to wild type marrow in contrast to the female setting where no significant differences were seen. As hormonal signals have been shown to regulate expression of HOX genes and differences in clonogenicity of male and female stem cells has been previously demonstrated, we reasoned this phenomenon could be secondary to extrinsic stimuli in vivo. The relatively uniform Hoxa11 levels in male and female knockout mice, however, suggested that cell intrinsic factors may also play a role. We therefore overexpressed HOXA11 into male and female wild type bone marrow ex vivo for colony forming assays to determine if elevated levels of the HOXA11 protein led to functional differences. This assay demonstrated a clear enhancement of self-renewal in male but not female bone marrow in contrast to HOXA9 overexpression which serially replated in both genders. Combined these data demonstrate that loss of function alterations in Hoxa11os transcripts lead to upregulation of Hoxa11 and gender specific hematopoietic progenitor cell perturbations. Ongoing efforts include competitive transplant studies as well as RNA and ChIP sequencing to identify gender specific downstream targets of Hoxa11 in the hematopoietic compartment in order to understand the selective expansion of progenitor subsets and male specific self-renewal capacity of this protein. These data will contribute to our understanding on how HOXA11-AS translocations promote oncogenesis. Disclosures Zwaan: Daiichi Sankyo: Consultancy; Sanofi: Consultancy; Roche: Consultancy; Pfizer: Research Funding; BMS: Research Funding; Incyte: Consultancy; Celgene: Consultancy, Research Funding; Servier: Consultancy; Jazz Pharmaceuticals: Other: Travel support; Janssen: Consultancy. Gruber:Bristol-Myers Squibb: Consultancy.

Small Molecule MIRA-1 Induces p53-Independent Apoptosis in Multiple Myeloma Cells Through Activation of the p38 MAPK Signaling Pathway.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2937-2937
Author(s):  
Manujendra N Saha ◽  
Hua Jiang ◽  
Yijun Yang ◽  
Donna Reece ◽  
Hong Chang
Keyword(s):  
Signaling Pathway ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Mapk Signaling ◽  
Mapk Signaling Pathway ◽  
Mutant P53 ◽  
Wild Type ◽  
Apoptotic Protein ◽  
Induced Apoptosis ◽  
Dose Dependent

Abstract Abstract 2937 Mutation of p53, a tumor suppressor protein, is relatively rare (∼10% in newly diagnosed patients) in multiple myeloma (MM). However, p53 mutations/deletions are important risk factors for predicting the resistant to chemotherapy and no treatment is currently available for this subgroup of patients. MIRA-1, a novel class of small molecules with the ability to restore wild type conformation and function to mutant p53, induces apoptosis in different types of solid tumors harboring mutant p53. However, its effect on MM cells is not known. In this study we examined the ability of MIRA-1 to induce cytotoxic and apoptotic response in MM cells and inhibit tumor growth in MM mouse xenograft model. In addition, we explored the molecular mechanisms of MIRA-1-induced apoptosis in MM cells. Treatment of MM cells with MIRA-1 resulted in a time- and dose-dependent decrease in survival and increase in apoptosis of MM cells harboring either wild type (MM.1S, H929) or mutant (U266, 8226, and LP1) p53 suggesting that MIRA-induced apoptosis in MM cells is independent of p53 status. The IC50 of MIRA-1 observed in these cells was ranged between 10 and 15 μM. In addition, MIRA-1 elicited a dose-dependent inhibition of myeloma cell growth in seven primary MM samples with an average IC50of 10 μM. Two of the seven patient samples harbors p53 mutations/deletions. In contrast, MIRA-1 did not have a significant inhibitory effect on the survival of bone marrow or peripheral blood mononuclear cells obtained from three healthy donors at the concentrations (10–20 μM) that induced apoptosis of MM cells, indicating a preferential killing of myeloma cells by this drug. Apoptosis induced by MIRA-1 in MM cells harbouring either wild type or mutant p53 was associated with time- and dose-dependent activation of caspas-8, caspase-3 and PARP with subsequent up-regulation of a pro-apoptotic protein, Noxa and down-regulation of an anti-apoptotic protein, Mcl-1. Interestingly, MIRA-1 did not significantly modulate the level of p53 expression, although immunoprecipitation studies confirmed the restoration of wild type conformation of mutant p53 in LP1 and 8226 cells. Importantly, genetic knockdown of p53 using siRNA against wild type or mutant p53 had only a little effect on apoptosis induction by MIRA-1 in MM.1S or LP1 cells, respectively, confirming that apoptosis induction by MIRA-1 in MM cells is independent of p53. Furthermore, the combination of MIRA-1 with current anti-myeloma agents, dexamethasone or doxorubicin displayed synergistic cytotoxic response in MM.1S or LP1 cells (CI<1; p<0.05). To delineate the molecular mechanisms of apoptosis in MM cells induced by MIRA-1, we performed RT2 profiler PCR array analysis for the differential expression of 84 genes related to mitogen activated protein kinase (MAPK) signaling pathway. A significant number of genes of the MAPK family including MAP3K: MAP3K2 (MEKK2), MAP3K4 (MEKK4), PAK1; MAP2K: MAP2K5 (MEK5); and MAPK: MAPK11 (p38bMAPK) as well as transcription factors such as c-Jun, c-FOS, EGR1, and MKNK1, whose expression is induced by MAPK signaling, were up-regulated by more than 2-fold in MIRA-1-treated 8226 cells. On the other hand, expression of the scaffolding/anchoring genes, MAPK8IP2 (JIP-1) was down-regulated by ∼2-fold. Up-regulations of c-Jun, c-Fos, and EGR1 at their protein levels were further confirmed by Western blot analysis of MM.1S and 8226 cells treated with MIRA-1. Importantly, Western blot analysis revealed that treatment of MIRA-1 resulted in a time- and dose-dependent increase of phosphorylated p38 MAPK level in both MM.1S and 8226 cells. Taken together, our data indicates that activation of the MAPK signaling pathway is, at least in part, associated with MIRA-1-induced apoptosis of MM cells. Finally, we evaluated anti-tumorigenic potential of MIRA-1 in MM xenograft SCID mouse models. 8266 cells were inoculated into SCID mice and the mice received i.p. injections of either 100 μL PBS (control) or 10 mg/kg MIRA-1 once daily for 18 days after tumor formation was evident. Administration of MIRA-1 resulted in significant inhibition of tumor growth (p<0.05) and increase in survival (p=0.007) of the mice with no apparent toxicity. Our study for the first time demonstrates potent in vitro and in vivo anti-myeloma activity of MIRA-1 and thus providing a framework for clinical evaluation of MIRA-1 either alone or in combination with current anti-myeloma agents. Disclosures: Reece: Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Millennium Pharmaceuticals: Research Funding.

Bone Marrow Stromal Cell-Derived Exosomes Facilitate Multiple Myeloma Cell Survival Through Inhibition Of The JNK Pathway

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 679-679
Author(s):  
Jinheng Wang ◽  
An Hendrix ◽  
Elke De Bruyne ◽  
Els Van Valckenborgh ◽  
Eddy Himpe ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Western Blot ◽  
Cell Viability ◽  
Cell Survival ◽  
Caspase 3 ◽  
Annexin V ◽  
Jnk Pathway ◽  
Functional Components

Abstract Interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells plays a crucial role in MM pathogenesis by secreting growth factors, cytokines, and other functional components. Exosomes are 30-100nm diameter membranous vesicles constitutively released by several cell types including reticulocytes, cytotoxic T lymphocytes, B lymphocytes, epithelial and endothelial cells. Exosomes mediate local cell-cell communication by transferring mRNAs, miRNAs and proteins. Due to their ability to transfer functional components, exosomes play multiple roles by stimulating target cells, transferring membrane receptors, delivering proteins, and inducing epigenetic changes in recipient cells. Although the promotion of MM growth and survival induced by BMSCs has been studied, the role of BMSC-derived exosomes in this action remains unclear. Here, we investigated the effect and mechanisms of BMSC-derived exosomes on the proliferation and survival of MM cells using the murine 5T33MM model. This model mimics the human disease closely and of this model two lines exist: the 5T33MMvv model which is propagated in vivo and the 5T33MMvt line which is derived from 5T33MMvv cells but which can grow stroma-independently. Exosomes were isolated from conditioned medium using the ExoQuick-TC Exosome Precipitation Solution (System Biosciences) after culture of primary BMSCs obtained from naïve C57BL/KaLwRij mice or 5T33MM diseased mice. The size of exosomes derived from naïve BMSCs, 5T33 BMSCs and 5T33MMvt cells were confirmed using a NanoSight LM10. Several exosomal markers such as CD63, Flotillin-1, heat shock protein 90 (HSP90), and HSP70 were detected using Western blot. We co-cultured the BMSCs or MM cells with fluorescent dye-labeled exosomes to examine whether exosomes could be transferred into cells. The results showed that both naïve and 5T33 BMSC-derived exosomes could fuse with 5T33MMvt cells and that the uptake of 5T33MMvt cell-derived exosomes by BMSCs was also observed. As several cytokines were found to be present in BMSC- and MMvt cell-derived exosomes, this suggests that BMSCs and MM cells could exchange cytokines with each other through exosomes secretion and uptake. Furthermore, the cytokine composition of 5T33BMSC-derived exosomes compared to naïve BMSC-derived exosomes was different. We next performed luminescent cell viability assays, BrdU cell proliferation assays and 7-AAD/annexin-V stainings to examine the effects of BMSC-derived exosomes on MM cell viability, proliferation and survival, respectively. Both naïve and 5T33 BMSC-derived exosomes increased 5T33MMvt and MMvv cell viability in a dose- and time-dependently manner. BrdU uptake in 5T33MMvt and MMvv cells was also increased after treatment with BMSC-derived exosomes. Significantly reduced apoptosis of 5T33 MMvt and MMvv cells was observed when they were treated with BMSC-derived exosomes as judged by 7-AAD/annexin-V staining. 5T33MMvt and MMvv cells were treated with different amounts of BMSC-derived exosomes and apoptosis-related proteins Bcl-2, Bax, and caspase-3 were determined using western blot. Bcl-2 was increased slightly and activated (cleaved) caspase-3 was reduced after co-culture with exosomes, coinciding with the results of 7-AAD/annexin-V staining. To elucidate the mechanisms responsible for exosome-induced MM cell survival, we examined the activation of several proteins involved. Reduced phosphorylation of p53, p38MAPK and JNK were detected when 5T33MMvt were treated with naïve-BMSC-derived exosomes for 24h, whereas phosphorylated Erk1/2, Akt, and IGF1Rβ were not changed. Surprisingly, activation of p53 and p38MAPK were not changed after the treatment with 5T33 BMSC-derived exosomes. 5T33 BMSC-derived exosomes further decreased the activation of JNK, Bim expression and phosphorylated Bim compared to naïve BMSC-derived exosomes. As Bim is a pro-apoptosis protein, mainly regulated by the JNK pathway; promotion of MM cell survival likely results from the inhibition of the JNK pathway by BMSC-derived exosomes. In summary, our results demonstrate a positive role for BMSC-derived exosomes in induction of MM cell proliferation and survival. BMSC-derived exosomes could inhibit the JNK pathway, thereby reducing caspase-3 activation and protecting MM cells from apoptosis. Disclosures: No relevant conflicts of interest to declare.

Sqstm1 Is Required to Retain Hematopoietic Stem Cell/ Progenitors As a Negative Regulator of Macrophage-Dependent Inflammatory Signaling in the Bone Marrow Osteoblastic Niche

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 350-350
Author(s):  
Kyung-Hee Chang ◽  
Amitava Sengupta ◽  
Ramesh C Nayak ◽  
Angeles Duran ◽  
Sang Jun Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Negative Regulator ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
P Retention

Abstract In the bone marrow (BM), hematopoietic stem cells and progenitors (HSC/P) reside in specific anatomical niches. Among these niches, a functional osteoblast (Ob)-macrophage (MΦ) niche has been described where Ob and MΦ (so called "osteomacs") are in direct relationship. A connection between innate immunity surveillance and traffic of hematopoietic stem cells/progenitors (HSC/P) has been demonstrated but the regulatory signals that instruct immune regulation from MΦ and Ob on HSC/P circulation are unknown. The adaptor protein sequestosome 1 (Sqstm1), contains a Phox bemp1 (PB1) domain which regulates signal specificities through PB1-PB1 scaffolding and processes of autophagy. Using microenvironment and osteoblast-specific mice deficient in Sqstm1, we discovered that the deficiency of Sqstm1 results in macrophage contact-dependent activation of Ob IKK/NF-κB, in vitro and in vivo repression of Ccl4 (a CCR5 binding chemokine that has been shown to modulate microenvironment Cxcl12-mediated responses of HSC/P), HSC/P egress and deficient BM homing of wild-type HSC/P. Interestingly, while Ccl4 expression is practically undetectable in wild-type or Sqstm1-/- Ob, primary Ob co-cultured with wild-type BM-derived MΦ strongly upregulate Ccl4 expression, which returns to normal levels upon genetic deletion of Ob Sqstm1. We discovered that MΦ can activate an inflammatory pathway in wild-type Ob which include upregulation of activated focal adhesion kinase (p-FAK), IκB kinase (IKK), nuclear factor (NF)-κB and Ccl4 expression through direct cell-to-cell interaction. Sqstm1-/- Ob cocultured with MΦ strongly upregulated p-IKBα and NF-κB activity, downregulated Ccl4 expression and secretion and repressed osteogenesis. Forced expression of Sqstm1, but not of an oligomerization-deficient mutant, in Sqstm1-/- Ob restored normal levels of p-IKBα, NF-κB activity, Ccl4 expression and osteogenic differentiation, indicating that Sqstm1 dependent Ccl4 expression depends on localization to the autophagosome formation site. Finally, Ob Sqstm1 deficiency results in upregulation of Nbr1, a protein containing a PB1 interacting domain. Combined deficiency of Sqstm1 and Nbr1 rescues all in vivo and in vitro phenotypes of Sqstm1 deficiency related to osteogenesis and HSC/P egression in vivo. Together, this data indicated that Sqstm1 oligomerization and functional repression of its PB1 binding partner Nbr1 are required for Ob dependent Ccl4 production and HSC/P retention, resulting in a functional signaling network affecting at least three cell types. A functional ‘MΦ-Ob niche’ is required for HSC/P retention where Ob Sqstm1 is a negative regulator of MΦ dependent Ob NF-κB activation, Ob differentiation and BM HSC/P traffic to circulation. Disclosures Starczynowski: Celgene: Research Funding. Cancelas:Cerus Co: Research Funding; P2D Inc: Employment; Terumo BCT: Research Funding; Haemonetics Inc: Research Funding; MacoPharma LLC: Research Funding; Therapure Inc.: Consultancy, Research Funding; Biomedical Excellence for Safer Transfusion: Research Funding; New Health Sciences Inc: Consultancy.

Ixazomib, Dexamethasone and Rituximab in Previously Untreated Patients with Waldenström Macroglobulinemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2956-2956 ◽  
Author(s):  
Jorge J. Castillo ◽  
Joshua Gustine ◽  
Kirsten Meid ◽  
Toni Dubeau ◽  
Guang Yang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Median Time ◽  
Research Funding ◽  
Response Rate ◽  
Bone Marrow Involvement ◽  
B Cell Lymphoma ◽  
Induction Phase ◽  
Primary Therapy ◽  
Wild Type ◽  
Median Serum

Abstract Introduction: Waldenström's macroglobulinemia (WM) is an incurable B-cell lymphoma characterized by the accumulation of IgM-secreting lymphoplasmacytic cells in the bone marrow and other organs. Bortezomib in combination with rituximab and dexamethasone (BDR) is highly active as primary therapy in WM, though treatment-related neuropathy is common with BDR in WM, and often leads to premature treatment discontinuation. Ixazomib is an orally administered proteasome inhibitor with limited neuropathy that is active in myeloma, but has not been previously evaluated in WM Methods: Symptomatic, previously untreated patients with a clinicopathological diagnosis of WM were included in this prospective, single-arm phase II study evaluating ixazomib 4 mg PO on days 1, 8 and 15 + dexamethasone 20 mg PO/IV on days 1, 8 and 15 + rituximab 375 mg/m2 IV on day 1 (IDR) were administered for six 4-week cycles (induction) followed by six 8-week cycles (maintenance). Rituximab was held for the first two cycles of therapy to minimize risk of IgM flare. Zoster prophylaxis and proton pump inhibitors were administered throughout IDR therapy. The study was approved by the institutional review board at the Dana-Farber Cancer Institute, and registered under Clinicaltrials.gov ID NCT02400437. Results: Twenty-six WM patients were enrolled and were exposed to IDR therapy. The median age at WM diagnosis was 63 years (range 46-81 years) and the median age at initiation of therapy was 65 years (range 46-82 years). Baseline median hemoglobin was 10.2 g/dl (range 6.9-13.2 g/dL), median serum IgM level was 5,068 mg/dl (range 653-7,650 mg/dL), 46% of patients had lymphadenopathy and 12% had splenomegaly. The median bone marrow involvement was 55% (range 5-95%). The MYD88 L265P gene mutation was identified in all cases. CXCR4 gene mutations were identified in 15 patients (58%), of whom 10 (67%) had nonsense, and 5 (33%) frameshift mutations. Sixteen patients have completed the induction phase of therapy at this time. Following induction therapy, the median serum IgM level decreased to 2,316 mg/dl (range 287-5,820 mg/dL), median hemoglobin increased to 13.1 mg/dl (range 10.4-14.6 g/dL), and median bone marrow involvement decreased to 23% (range 0-76%). P-value <0.001 for all comparisons against baseline. Using consensus response criteria, the overall response rate was 88% (VGPR 6%, PR 44%, MR 38%) with a major response rate of 50%. Major responses (VGPR + PR) were observed in 47% of patients with CXCR4 mutations versus 64% in those who were wild-type CXCR4 (p=0.32). The median time to response was 8 weeks. The median time to response in CXCR4 mutant patients was 12 weeks versus 8 weeks in wild-type CXCR4 patients (log-rank p=0.03). Four patients have been taken off study; 2 for lack of response, 1 due to lack of clinical benefit with persistent failure to thrive while in PR, and 1 for progressive neuropathy while in PR although in part due to worsening of diabetic neuropathy. No other grade 3 or 4 adverse events were reported. Conclusion: These preliminary data suggest that the combination of IDR is an active and well-tolerated, neuropathy-sparing regimen in symptomatic untreated WM patients. Disclosures Castillo: Biogen: Consultancy; Otsuka: Consultancy; Abbvie: Research Funding; Pharmacyclics: Honoraria; Millennium: Research Funding; Janssen: Honoraria.

scholarly journals Targeting Wild-Type and Mutant p53 with Small Molecule CP-31398 Blocks the Growth of Rhabdomyosarcoma by Inducing Reactive Oxygen Species–Dependent Apoptosis

2010 ◽  
Vol 70 (16) ◽  
pp. 6566-6576 ◽  
Author(s):  
Jianmin Xu ◽  
Laura Timares ◽  
Clay Heilpern ◽  
Zhiping Weng ◽  
Changzhao Li ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Small Molecule ◽  
Reactive Oxygen ◽  
Mutant P53 ◽  
Oxygen Species ◽  
Wild Type

scholarly journals ASXL2 Is Recurrently Mutated in t(8;21) AML and Regulates Hematopoietic Development

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1051-1051
Author(s):  
Vikas Madan ◽  
Lin Han ◽  
Norimichi Hattori ◽  
Anand Mayakonda ◽  
Qiao-Yang Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Research Funding ◽  
Hematopoietic Differentiation ◽  
Wild Type ◽  
Flow Cytometric ◽  
Deficient Mice

Abstract Chromosomal translocation t(8;21) (q22;q22) leading to generation of oncogenic RUNX1-RUNX1T1 fusion is a cytogenetic abnormality observed in about 10% of acute myelogenous leukemia (AML). Studies in animal models and recent next generation sequencing approaches have suggested cooperativity of secondary genetic lesions with t(8;21) in inducing leukemogenesis. In this study, we used targeted and whole exome sequencing of 93 cases (including 30 with matched relapse samples) to profile the mutational landscape of t(8;21) AML at initial diagnosis and post-therapy relapse. We identified recurrent mutations of KIT, TET2, MGA, FLT3, NRAS, DHX15, ASXL1 and KMT2Dgenes in this subtype of AML. In addition, high frequency of truncating alterations in ASXL2 gene (19%) also occurred in our cohort. ASXL2 is a member of mammalian ASXL family involved in epigenetic regulation through recruitment of polycomb or trithorax complexes. Unlike its closely related homolog ASXL1, which is mutated in several hematological malignancies including AML, MDS, MPN and others; mutations of ASXL2 occur specifically in t(8;21) AML. We observed that lentiviral shRNA-mediated silencing of ASXL2 impaired in vitro differentiation of t(8;21) AML cell line, Kasumi-1, and enhanced its colony forming ability. Gene expression analysis uncovered dysregulated expression of several key hematopoiesis genes such as IKZF2, JAG1, TAL1 and ARID5B in ASXL2 knockdown Kasumi-1 cells. Further, to investigate implications of loss of ASXL2 in vivo, we examined hematopoiesis in Asxl2 deficient mice. We observed an age-dependent increase in white blood cell count in the peripheral blood of Asxl2 KO mice. Myeloid progenitors from Asxl2 deficient mice possessed higher re-plating ability and displayed altered differentiation potential in vitro. Flow cytometric analysis of >1 year old mice revealed increased proportion of Lin-Sca1+Kit+ (LSK) cells in the bone marrow of Asxl2 deficient mice, while the overall bone marrow cellularity was significantly reduced. In vivo 5-bromo-2'-deoxyuridine incorporation assay showed increased cycling of LSK cells in mice lacking Asxl2. Asxl2 deficiency also led to perturbed maturation of myeloid and erythroid precursors in the bone marrow, which resulted in altered proportions of mature myeloid populations in spleen and peripheral blood. Further, splenomegaly was observed in old ASXL2 KO mice and histological and flow cytometric examination of ASXL2 deficient spleens demonstrated increased extramedullary hematopoiesis and myeloproliferation compared with the wild-type controls. Surprisingly, loss of ASXL2 also led to impaired T cell development as indicated by severe block in maturation of CD4-CD8- double negative (DN) population in mice >1 year old. These findings established a critical role of Asxl2 in maintaining steady state hematopoiesis. To gain mechanistic insights into its role during hematopoietic differentiation, we investigated changes in histone marks and gene expression affected by loss of Asxl2. Whole transcriptome sequencing of LSK population revealed dysregulated expression of key myeloid-specific genes including Mpo, Ltf, Ngp Ctsg, Camp and Csf1rin cells lacking Asxl2 compared to wild-type control. Asxl2 deficiency also caused changes in histone modifications, specifically H3K27 trimethylation levels were decreased and H2AK119 ubiquitination levels were increased in Asxl2 KO bone marrow cells. Global changes in histone marks in control and Asxl2 deficient mice are being investigated using ChIP-Sequencing. Finally, to examine cooperativity between the loss of Asxl2 and RUNX1-RUNX1T1 in leukemogenesis, KO and wild-type fetal liver cells were transduced with retrovirus expressing AML1-ETO 9a oncogene and transplanted into irradiated recipient mice, the results of this ongoing study will be discussed. Overall, our sequencing studies have identified ASXL2 as a gene frequently altered in t(8;21) AML. Functional studies in mouse model reveal that loss of ASXL2 causes defects in hematopoietic differentiation and leads to myeloproliferation, suggesting an essential role of ASXL2 in normal and malignant hematopoiesis. *LH and NH contributed equally Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

scholarly journals An Open-Label Phase I/IIa Study to Evaluate the Safety and Efficacy of CCS1477, a First in Clinic Inhibitor of the p300/CPB Bromodomains, As Monotherapy in Patients with Advanced Haematological Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1266-1266 ◽  
Author(s):  
Tomasz Knurowski ◽  
Karen Clegg ◽  
Nigel Brooks ◽  
Fay Ashby ◽  
Neil A Pegg ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Board Of Directors ◽  
Research Funding ◽  
Haematological Malignancies ◽  
Loss Of Function ◽  
Employment Equity ◽  
Advisory Committees ◽  
Measurable Disease ◽  
Equity Ownership

Background CCS1477 is a first in class potent, selective and orally bioavailable inhibitor of the bromodomains of p300 and CBP, two closely related histone acetyl transferases with oncogenic roles in haematological malignancies. In pre-clinical studies, CCS1477 was found to be a potent inhibitor of cell proliferation in acute myeloid leukaemia (AML) multiple myeloma (MM) and non-Hodgkin lymphoma (NHL) cell lines. In primary patient AML blast cells CCS1477 inhibited proliferation through a combination of cell cycle arrest at the G1/S transition and an induction of differentiation (up-regulation of CD11b and CD86). CCS1477 has significant anti-tumour activity, inducing tumour regressions in xenograft models of AML and MM. These effects were accompanied by significant reductions in tumour MYC and IRF4 expression. Additionally, there are molecular features of certain haematological malignancies that are likely to increase the sensitivity to p300/CBP inhibition with CCS1477. For example, in B-cell lymphomas there are frequent loss of function mutations in CBP that are associated with heightened sensitivity to pre-clinical inhibition of corresponding non-mutated p300. CCS1477 represents a novel and differentiated approach to inhibiting cell proliferation and survival and offers a potential new therapeutic option for patients who have relapsed or are refractory to current standard of care therapies in AML, MM or NHL. Study Design and Methods This study is the first time that CCS1477 is being dosed in patients with haematological malignancies. The Phase I/IIa study aims to determine the maximum tolerated dose (MTD) and/or recommended Phase II dose (RP2D) and schedule(s) of CCS1477 and investigate clinical activity of CCS1477 monotherapy in patients with haematological malignancies. This study will also characterise the pharmacokinetics (PK) of CCS1477 and explore potential biological activity by assessing pharmacodynamic and exploratory biomarkers. The trial aims to enrol approximately 90 patients and is currently recruiting in the UK with plans to open additional sites in the USA. Key inclusion criteria include patients with confirmed (per standard disease specific diagnostic criteria), relapsed or refractory haematological malignancies (AML, MM and NHL). Patients must have received standard therapy which for the majority of therapeutic indications is at least 2 prior lines of therapy. Single dose and steady state pharmacokinetics will be determined in all patients. AML response will be measured in bone marrow samples. Myeloma response will be evaluated according to the 'International Myeloma Working Group Response Criteria' based on changes in M protein in blood and/or urine, changes in serum free light chains if measurable, and changes on imaging and/or bone marrow if applicable and according to the guidelines. In NHL patients, tumour assessments will be done for measurable disease, non-measurable disease, and new lesions on CT (or magnetic resonance imaging [MRI]) and/or combined with visual assessment of [18F]2-fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET) for response assessment per recent International Working Group consensus criteria (RECIL 2017), until progression The study will begin with two parallel monotherapy dose-escalation arms; Arm 1: Relapsed or refractory NHL and MM; Arm2: Relapsed or refractory AML/high-risk MDS. Once a recommended phase 2 dose/schedule is reached, three monotherapy expansion arms will be opened in AML/high-risk MDS (15 patients), MM (15 patients) and NHL (30 patients). Blood samples along with bone marrow biopsies and aspirates will be collected for exploratory biomarker analysis to understand mechanisms of response to treatment or disease progression. This will include the analysis of tumour-specific and circulating biomarkers, such as tumour DNA, mRNA, proteins or metabolites. In NHL patients, analysis of CBP (and p300) mutations will be undertaken to allow retrospective correlation with tumour response and to determine if loss of function mutations in the genes for either proteins can be utilised as response predictive biomarkers in future studies. Disclosures Clegg: CellCentric Ltd: Employment, Equity Ownership. Brooks:CellCentric Ltd: Employment, Equity Ownership. Ashby:CellCentric Ltd: Employment, Equity Ownership. Pegg:CellCentric Ltd: Employment, Equity Ownership. West:CellCentric Ltd: Employment, Equity Ownership. Somervaille:Novartis: Consultancy. Knapper:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Honoraria; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Tolero: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Davies:ADCT Therapeutics: Honoraria, Research Funding; MorphoSys AG: Honoraria, Membership on an entity's Board of Directors or advisory committees; BioInvent: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Karyopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Honoraria, Research Funding; GSK: Research Funding; Pfizer: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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