Disruption of MDM2-p53 Interactions by a Small Molecule Inhibitor RG7112 Increases Apoptosis and Impairs Polyploidization of Human Megakaryocytes

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1082-1082 ◽  
Author(s):  
Camelia Iancu-Rubin ◽  
Mosoyan Goar ◽  
Ronald Hoffman
Keyword(s):  
Cell Cycle ◽  
Small Molecule ◽  
Research Funding ◽  
Phase I Clinical Trials ◽  
Viable Cells ◽  
Inactive Form ◽  
Cd34 Cells ◽  
P53 Activation

Abstract Abstract 1082 Megakaryocyte (MK) development is characterized by polyploidization, cytoplasmic maturation and proplatelet formation, which culminates in the release of platelets into the circulation. The tumor suppressor p53 plays a critical role in the regulation of both cell cycle and apoptosis; its function is tightly controlled by the murine double minute (MDM2) protein which facilitates p53 degradation and inhibits p53 transcriptional activity. MK ploidy results from a disruption of normal cell cycle progression termed endomitosis while platelet release is believed to depend on apoptotic processes. The role of p53-MDM2 in MK in these two processes has not been clearly defined. A small molecule RG7112, which disrupts MDM2-p53 interaction, has shown promising anti-tumor effects in phase I clinical trials. This beneficial outcome has, however, been associated with the development of thrombocytopenia. We, therefore, used RG7112 as pharmacological probe to examine the effects of disruption of the MDM2-p53 regulatory loop on MK. We determined the effects of RG7112 on primary human MK by utilizing an in vitro system in which MK were generated from BM-derived CD34+ cells. We first demonstrated that both p53 and MDM2 transcripts are up-regulated as MK differentiation progresses. The ability of CD34+ cells to proliferate in the absence or presence of various concentrations of RG7112 was then evaluated both in liquid cultures and in CFU-MK colony assays. CD34+ cells exposed to 10 μM RG7112 for 7 days generated 70% fewer viable cells as compared to control cells exposed to the inactive form of the drug (p value = 0.0038). Furthermore, CD34+ cells treated with RG7112 formed up to 40% less CFU-MK colonies as compared to untreated cells. An assessment of apoptosis of MK precursors generated in the presence of RG7112 revealed that 69.5+2.1% were Annexin V positive as compared to 31.5+3.5% present in control cultures. These findings are consistent with the previously reported role of RG7112 in inducing p53 activation and apoptosis. Interestingly, phenotypical characterization of the viable cells generated under identical culture conditions, showed that RG7112 treatment did not interfere with the ability of CD34+ cells to acquire markers of MK differentiation during the first 7 days of culture since similar degrees of CD41 and CD42 expression were observed in the absence and in the presence of the drug. Likewise, exposure of MK precursors to the drug for 7 additional days (i.e. later stages of maturation) did not influence CD41 and CD42 expression. By contrast, cells differentiated in the presence of 5 μM RG7112 generated 50% fewer polyploid MK with greater than 4N DNA content as compared to those treated with the inactive form of the drug. Moreover, the negative effects on ploidy were associated with p53 activation, as assessed by the increased levels of p21 protein, a direct target of p53 which is known to limit polyploidization of primary MK. Finally, platelets generated in vitro were analyzed phenotypically and quantitated by dual labeling with anti-CD41 antibodies and thiazole orange (TO). The number of CD41+/TO+platelets derived from MK generated in the presence of RG7112 was reduced by 22% as compared to control. Based on these findings, we conclude that RG7112 impacts megakaryopoiesis by two potential mechanisms: 1) Impairing the ability of CD34+ cells to generate MK precursors due to increased apoptosis; 2) Limiting polyploidization during the late stages of development due to phamacological activation of p53. A combination of these two effects may provide an explanation for thrombocytopenia observed in patients receiving this drug and suggests that p53 plays an important role in normal human thrombocytopoiesis. Disclosures: Iancu-Rubin: Roche: Research Funding. Hoffman:Roche: Research Funding.

scholarly journals Combination of BCR-ABL1 Tyrosine Kinase Inhibitors with a Small Molecule LIM kinase1/2 Inhibitor in BCR-ABL1 Positive Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2705-2705
Author(s):  
Thorsten Braun ◽  
Jeannig Berrou ◽  
Renaud Prudent ◽  
Hanane Djamai ◽  
Melanie Dupont ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Introduction: LIM kinases 1 and 2 (LIMK1/2) are downstream effectors at the crossroads of different signaling pathways implicated in the dynamics of the cytoskeleton via phosphorylation of cofilin family proteins, degradation of the matrix by phosphorylating MT1-MMP and control of the activity of Aurora kinase A. Recently, the oncogenic role of Rho kinases (ROCK) was identified to be constitutively activated by BCR-ABL1, FLT3-ITD and KIT in hematologic malignancies via PI3 kinase and Rho GTPase mediated phosphorylation. Upon activation, ROCK phosphorylates LIMK1/2 leading to inactivation of cofilin by its phosphorylation and polymerization of actin and microtubules and possibly to other biological effects mediated by LIMK1/2, not yet fully understood. Here, we demonstrate synergy of a LIMK1/2 inhibitor with BCR-ABL1 tyrosine kinase inhibitors (TKI) in vitro and in vivo in different models for BCR-ABL1 driven ALL. Materials and Methods: Expression of LIMK1/2 was determined by RT-qPCR and WB in cell lines. Phosphorylation of cofilin was detected by WB. A small molecule inhibitor of LIMK1/2 was tested alone and in combination with imatinib, dasatinib, nilotinib and ponatinib in BCR-ABL1 positive ALL cell lines TOM-1 and BV-173. Cell viability and IC50 was assessed by MTS assays after exposure to LIMK1/2 inhibitor for 72h. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTS assays and combination index (CI) values were calculated according to the Chou-Talalay model. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) DNA intercalation. Induction of apoptosis was evaluated by annexin-V exposure and PI incorporation at 72h with increasing doses of LIMK1/2 inhibitor. Peripheral blood (PB) nucleated cells from apharesis products of healthy donors obtained after informed consent according to Helsinki declaration were incubated with or without LIMK1/2 inhibitor for 72h, and then enriched for CD34+ cells by immuno-magnetic selection and seeded in triplicate in methylcellulose FCS and cytokines. In vivo experiments were performed in C57Bl/6 mice injected with BCR-ABL-induced B-ALL cells. These were obtained by transduction of CDKN2A-deficient B-cell progenitors with a retrovirus coding for BCR-ABL1 (P185) and GFP, followed by transplantation in sub-lethally-irradiated recipient C57Bl/6 mice. Mice were treated either with LIMK1/2 inhibitor, nilotinib or the combination of both and compared to untreated control mice. Results: Expression of the two isoforms LIMK1 and LIMK2 in TOM-1 and BV-173 cells could be detected by RT-qPCR and at the protein level by WB. IC50 after LIMK1/2 inhibitor exposure alone was 580nM in TOM-1 cells and 1000nM in BV-173 cells. All combination experiments with the LIMK1/2 inhibitor and imatinib, dasatinib, nilotinib and ponatinib yielded synergistic CI for treatment of both TOM-1 and BV-173 cell lines. Cell cycle arrest in the G1/S transition was detected and LIMK1/2 inhibition induced dose dependent apoptosis in TOM-1 and BV-173 cells up to 40% at doses <1000nM. Upon treatment with the LIMK1/2 inhibitor, decrease of LIMK1 protein expression could be detected by WB, while LIMK2 expression was left unaffected. In both cell lines, LIMK1/2 inhibitor exposure lead to activating downstream dephosphorylation of cofilin as expected. No significant toxicity of increasing doses of LIMK1/2 inhibitor after exposure of CD34+ cells from healthy donors could be detected. To test the in vivo activity of LIMK1/2 inhibition, C57Bl/6 mice were transplanted with CDKN2Ako/BCR-ABL1+ B-ALL cells. Leukemic mice were treated with LIMK1/2 inhibitor alone, nilotinib or combination of LIMK1/2 inhibitor and nilotinib compared to untreated mice. The combination of nilotinib and LIMK1/2 inhibitor significantly delayed the appearance of leukemic cells in PB as detected by GFP+ cells once weekly or at death if possible with mice considered having leukemia if >1% GFP+ cells were detected in PB. Furthermore, nilotinib+LIMK1/2 inhibitor prolonged significantly the survival of mice compared to either nilotinib (p=0.0006) or LIMK1/2 inhibitor alone and untreated mice (p<0.0001) (Figure 1). Conclusion: Combination of LIMK1/2 inhibitor with BCR-ABL targeting TKI is synergistic and has significant anti-leukemic activity in BCR-ABL1+ ALL in vitro and in vivo models. Disclosures Braun: CELLIPSE: Research Funding. Prudent:CELLIPSE: Employment. Paublant:CELLIPSE: Employment. Baruchel:Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Shire: Research Funding; Servier: Consultancy; Amgen: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Roche: Consultancy. Dombret:CELLIPSE: Research Funding.

scholarly journals The APC/C Coactivator Cdh1 Controls Self-Renewal and Differentiation of Human and Murine HSPCs

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2650-2650
Author(s):  
Daniel Ewerth ◽  
Stefanie Kreutmair ◽  
Andrea Schmidts ◽  
Marie Follo ◽  
Dagmar Wider ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
Research Funding ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Cd34 Cells

Abstract Introduction: The balance between differentiation and self-renewal in hematopoietic stem and progenitor cells (HSPCs) is crucial for homeostasis and lifelong blood cell production. Differentiation is predominantly initiated in the G1 phase of the cell cycle when the E3 ligase anaphase-promoting complex or cyclosome (APC/C) is highly active. Its coactivator Cdh1 determines substrate specificity and mediates proteasomal degradation. Relevant target proteins are associated with cell fate decisions in G1/G0, and there is growing evidence that Cdh1 is an important regulator of differentiation. While this has already been demonstrated in neurons, muscle cells or osteoblasts, little is known about the role of APC/CCdh1 in hematopoiesis. Here we report on the function of Cdh1 in human and murine HSPCs in vitro and in vivo. Methods: Human CD34+ cells from the peripheral blood of G-CSF mobilized donors were exposed to different cytokine combinations and gains or losses of surface marker expression during cell division were determined. By using the established culture conditions Cdh1 expression was detected in distinct hematopoietic lineages and developmental states. CD34+ cells were transduced with a lentivirus to deplete Cdh1 by stably expressing shRNA and was then used for in vitro differentiation in liquid culture or CFU assay. In a second miR-based RNAi approach murine BM cells were depleted of Cdh1 and used for competitive transplantation assays. Complementary xenotransplantation of human Cdh1-depleted CD34+cells was carried out with NSG mice. Results: The stimulation of freshly thawed CD34+ cells with cytokines led to cell cycle entry and proliferation. Self-renewing cells preserved CD34 expression for up to 7 cell divisions with a low proliferation rate. In contrast, during granulopoiesis and erythropoiesis cells divided more frequently with rapid down-regulation of CD34. Cdh1 expression was tightly connected to differentiation status and proliferation properties. In vitro cultured CD34+ cellsand those from BM of healthy human donors showed the highest Cdh1 level compared to moderate or low expression in lymphoid and myeloid cells. Cdh1 is highly expressed at the transcriptional and translational level during both self-renewal and also when cells were directed toward erythroid differentiation. Therefore, high Cdh1 expression is characteristic of immature hematopoietic cells and differentiating precursors. The knockdown of Cdh1 (Cdh1-kd) did not affect proliferation or viability as detected by CFSE staining and measuring the cell cycle length via live-cell imaging. However, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with a lower frequency of glycophorin A+ cells. The functional relevance of Cdh1 depletion was verified in CFU assays. Cells with Cdh1-kd formed fewer primary colonies but significantly more secondary colonies, indicating a preference for self-renewal over differentiation. After competitive transplantation Cdh1-depleted murine BM cells showed a significant enhancement in the repopulation of PB, BM and spleen at week 3, while there was no change in cell cycle properties. However, after 8 weeks chimerism in each of the compartments was reduced to that of the control cells. Accordingly, higher LK and LSK frequencies supported the engraftment of Cdh1-depleted cells at week 3, but there was a significant decrease at week 8 compared to control cells, suggestive of stem cell exhaustion. The Cdh1 level also affected cell differentiation in vivo. After 8 weeks the population of B cells (B220+) was increased in transplanted Cdh1-kd cells and the frequency of mature granulocytes (CD11b+ Gr1high) was reduced. Consistently, human Cdh1-depleted CD34+ cells engrafted to a much higher degree in the murine BM 8 and 12 weeks after xenotransplantation, as shown by a higher frequency of human CD45+ cells. Moreover, the increase of human CD19+ B cells with Cdh1-kd confirmed the results of the competitive transplantation. Conclusions: Loss of the APC/C coactivator Cdh1 supports repopulation of murine HSPCs after transplantation with a lymphoid-biased differentiation, and was confirmed in xenotranplantation experiments. In the long-term, Cdh1 loss led to exhaustion of primitive LK and LSK population, highlighting the role of Cdh1 as a critical regulator of HSPC self-renewal and differentiation. Disclosures Engelhardt: Janssen: Research Funding; Amgen: Research Funding; MSD: Research Funding; Celgene: Research Funding.

Self-Renewal and Differentiation in Hematopoietic Stem and Progenitor Cells Is Controlled By the APC/C Coactivator Cdh1

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2370-2370
Author(s):  
Daniel Ewerth ◽  
Stefanie Kreutmair ◽  
Birgit Kügelgen ◽  
Dagmar Wider ◽  
Julia Felthaus ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Research Funding ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Introduction: Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously renew the hematopoietic system by differentiation into mature blood cells. The process of differentiation is predominantly initiated in G1 phase of the cell cycle when stem cells leave their quiescent state. During G1 the anaphase-promoting complex or cyclosome (APC/C) associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate proliferation. In addition, Cdh1 has been shown to control terminal differentiation in neurons, muscle cells or osteoblasts. Here we show that Cdh1 is also a critical regulator of human HSPC differentiation and self-renewal. Methods: Human CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokine combinations. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with flow cytometric detection of CD34 expression. The knockdown and overexpression of Cdh1 was achieved by lentiviral delivery of suitable vectors into target cells. After cell sorting transduced (GFP+) CD34+ cells were used for in vitro differentiation in liquid culture or CFU assay. For in vivo experiments purified cells were transplanted into NSG mice. Results: G-CSF mobilized CD34+ cells showed effective differentiation into granulocytes (SCF, G-CSF), erythrocytes (SCF, EPO) or extended self-renewal (SCF, TPO, Flt3-L) when stimulated in vitro. The differentiation was characterized by a fast downregulation of Cdh1 on protein level, while Cdh1 remained expressed under self-renewal conditions. A detailed analysis of different subsets, both in vitro and in vivo, showed high Cdh1 level in CD34+ cells and low expression in myeloid cells. Analysis of proliferation revealed lowest division rates during self-renewal, accompanied by higher frequency of CD34+ cells. The fastest proliferation was found after induction of erythropoiesis. These experiments also showed a more rapid decrease of HSPCs' colony-forming ability and of CD34+ cells during granulopoiesis after 2-3 cell divisions in contrast to a moderate decline under self-renewal conditions. The depletion of Cdh1 (Cdh1-kd) had no effect on total cell numbers or proliferation detected by CFSE during differentiation and self-renewal, but showed an increase in S phase cells. These results were confirmed at the single cell level by measuring the cell cycle length of individual cells. Independent of cell cycle regulation, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with lower frequency of Glycophorin A+ cells. In CFU assays, the Cdh1-kd resulted in less primary colony formation, notably CFU-GM and BFU-E, but significantly more secondary colonies compared to control cells. These results suggest that the majority of cells reside in a more undifferentiated state due to Cdh1-kd. The overexpression of Cdh1 showed reversed results with less S phase cells and tendency to increased differentiation in liquid culture and CFU assays. To further validate our results in vivo, we have established a NSG xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Preliminary data in murine hematopoiesis support our hypothesis showing enhanced PB chimerism upon Cdh1-kd. Looking for a mediator of these effects, we found the Cdh1 target protein TRRAP, a cofactor of many HAT complexes, increased upon Cdh1-kd under self-renewal conditions. We use currently RT-qPCR to determine, if this is caused by a transcriptional or post-translational mechanism. Conclusions: Loss of the APC/C coactivator Cdh1 supports self-renewal of CD34+ cells, represses erythropoiesis in vitro and facilitates engraftment capacity and B cell development of human HSPCs in vivo. This work was supported by Josè Carreras Leukemia Foundation grant DCJLS R10/14 (to ME+RW) Disclosures Ewerth: Josè Carreras Leukemia Foundation: Research Funding. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

Role of mTOR in Hematopoiesis and Hematopoietic Stem Cell Regulation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1490-1490
Author(s):  
Fukun Guo ◽  
Wei Liu ◽  
Kankana Chava ◽  
Jose Cancelas ◽  
George Thomas ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Stem Cell ◽  
Blood Cell ◽  
Cell Survival ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem

Abstract Abstract 1490 Poster Board I-513 The mammalian target of rapamycin (mTOR) integrates nutrients, growth factors, and cellular energy status to control protein synthesis that determines cell growth and metabolism. It is also known that mTOR plays an essential role in cell survival by regulating Akt/PKB signaling. By using the inhibitor rapamycin, mTOR has previously been suggested to regulate proliferation of megakaryocyte progenitors and late stage of megakaryocyte differentiation without a general impact on normal hematopoiesis or hematopoietic stem cell (HSC) function. Due to limitations of rapamycin and the early lethality of conventional mTOR gene targeted mice, the physiological role of mTOR in blood development remains undefined. In this study, we have utilized an inducible conditional mTOR knockout mouse model by crossbreeding mTORflox/flox mice with Mx-Cre mice that allow interferon-induced mTOR deletion in the bone marrow following a transplantation and polyI:C induction protocol, in an effort to determine the genetic role of mTOR in hematopoiesis. Depletion of mTOR drastically affected hematopoiesis in a blood cell autonomous manner in Mx-Cre;mTORflox/flox bone marrow transplant recipients: the mice showed marked reduction in BM cellularity and in the numbers of myeloid and lymphoid lineage cells, erythrocytes, and platelets in peripheral blood, bone marrow, and thymus, leading to bone marrow failure, blood cell exhaustion and lethality. In vitro colony-forming activities by bone marrow or spleen progenitors were completely abolished in the absence of mTOR. Interestingly, the number and frequency of HSCs in bone marrow (Lin−Sca-1+c-Kit+) increased transiently while the number of early progenitors (CMP, GMP, MEP, CLP) detected by cell surface markers remained unchanged or only mildly affected in the mutant mice within 14 days after polyI:C treatment. Concomitantly, mTOR deletion led to a massive egress of HSCs from bone marrow to distal organs including spleen (∼60-fold increase). Transplantation of mTOR−/− bone marrow cells into NOD-SCID mice or competitive transplantation of mTOR−/− bone marrow cells into BoyJ mice further demonstrated that mTOR deficiency caused a complete failure in HSC engraftment and repopulation. Surprisingly, at the cellular level these phenotypes are associated with increased proliferation of HSCs in vivo and in vitro by 60% and 2.5-fold, respectively, as assessed by 5-bromodeoxyuridine incorporation assays whereas the cell survival index appears to be unaffected. Moreover, mTOR−/− HSCs and progenitor cells displayed impaired adhesion to fibronectin CH296 fragment (∼30% decrease) and migration toward SDF-1α gradients (∼30% decrease). At the molecular level, gene chip microarray analysis of mTOR−/− HSCs revealed that the cell cycle regulators myb, wee1, FANCD2, and FANCE were significantly downregulated while Rb and E2F5 were upregulated, the survival/apoptosis regulators MCL1 and BCL2L1 were upregulated, and the actin cytoskeleton and cell extracellular matrix adhesion regulators Arp2/3 complex subunit 5, paxillin, laminin α5, integrin β3, and myosin light chain 6B were upregulated. Further, immunoblotting analysis of isolated Lin− cells showed that SCF-stimulated activation of translational regulators S6K and 4E-BP and survival regulator Akt were abolished upon mTOR deletion. Taken together, these data suggest that mTOR is a critical regulator of HSC quiescence, self-renewal, and engraftment through the regulation of cell cycle, survival and actin cytoskeleton signals, and is essential in multiple stages of hematopoiesis. Disclosures Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

Mimicking the Myeloma Niche: A 3D Bone-Derived Co-Culture System to Selectively Assess Bystander BMSCs and to Perform High-Throughput Drug Screening in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1822-1822
Author(s):  
Johannes M. Waldschmidt ◽  
Dagmar Wider ◽  
Andreas R. Thomsen ◽  
Claudius Klein ◽  
Christine Aldrian ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
High Throughput ◽  
Research Funding ◽  
Ex Vivo ◽  
Bm Niche ◽  
Culture Model ◽  
Bystander Cells

Abstract Introduction: Novel substances such as the next generation IMiD pomalidomide or the recently approved next generation proteasome inhibitor carfilzomib (Cfz) have considerably expanded our treatment options in MM, both of them influencing multiple myeloma (MM) interaction with bone marrow stroma cells (BMSCs), that provides an interesting target for anti MM therapy. More compounds directed at this disease critical crosstalk are currently under investigation, however the development of novel drugs remains inefficient, displayed by a substantial drop out rate of the 376 preclinical single agents tested since 1961 (Rongvaux, Annu Rev Immunol. 2013; Schüler, Expert Opin Biol Ther. 2013; Kortüm, CLML 2014). Our focus in the projected presented here was to develop a novel bone-derived in vitro 3D co-culture model specifically adapted to mimic the BM niche to more closely study the role of bone and BM bystander cells and to perform more reliable ex vivo compound screening in MM. Methods: Previous 2D models were compared to a novel 3D co-culture model (agarose matrix interlayer, 100 microwells/cm², 1.5mm in depth, permeable for oxygen+cytokines, but not for BMSCs utilizing U266, RPMI-8226, OPM-2 and primary BM patient (pt) cells, with and without HS-5 vs. M210B4 stroma support (Fig. A + Fig. B.a. for pt characteristics). Analyses covered Trypan Blue, Annexin/PI, MTT, FACS, cell cycle analyses and H2B-mCherry/cytochrome c-GFP assays (Udi, Br J Hematol. 2013). In a next step, primary bone-derived stroma cells were acquired from bones of C57BL/6 J mice. Bones were flushed, digested and FACS sorted in order to acquire single BM and bone bystander cell subtypes (MSPCs [mesenchymal stem and progenitor cells], endothelial cells, osteoblasts, PAS [PDFGRalphaSca1] and CaRs [CXCL12-abundant reticular cells]) which were then compared to HS-5 and M210B4 with regard to growth support, cytokine secretion and protection from anti-MM substances. Results: MMCLs and pt specimens were cultured at different concentrations (10 vs. 100 cells per microwell) with and without M210B4 demonstrating a growth advantage with vs. without M210B4 (Fig. B.b). Liquid overlay technique allowed cluster formation of pt specimens leading to more reliable propagation of pt material for up to 20d of culture. Apoptotic changes were assessed by confocal microscopy of RPMI8226 co-expressing fluorescently labelled histone 2B-mCherry (red) and cytochrome c-GFP (green) as indicators of late and early apoptosis. Comparing BMSCLs with regard to their MM growth support capacities, human HS-5 proved even more beneficent than M210B4 stroma (Fig. B.b). Phenotypic analyses of pt specimens showed decreased CXCR4 expression with vs. without BMSCs suggesting a dynamic regulation of homing molecules. The model was then used as an ex-vivo platform allowing both cytotoxicity and cell cycle analyses for the combination of bortezomib (Btz) vs. Cfz with ARRY-520 (kinesin spindle protein inhibitor). Btz (10nM) and Cfz (20nM) proved significantly cytotoxic compared to the control (U266 and pt specimens, respectively) after 48h of single agent treatment Fig.B.c). Compared to Btz (10nM, B10) and Cfz (20nM, C20) as single agents, the additional combination with ARRY-520 showed stronger additive cytotoxicity for Btz (A5+B10, median: 37.5% vs. 13.1%) than for Cfz (A5+C20, 38.6% vs. 34.1%). To note, the model could also be utilized for more profound analyses as depicted for G2/M cell cycle studies in Fig.B.d. 5nM ARRY-520 (A5) led to significant accumulation of OPM-2 cells in G2/M arrest after 48h of treatment confirming prior analyses (Hernández-Garcia Blood Suppl 4710,2014; Fig. B.d). Co-culture studies with different subsets of BM and bone-derived bystander cells are currently ongoing and will be presented at the meeting (Fig. B.e). Conclusions: More complex, 3D bone-derived high-throughput in vitro models are urgently needed to better predict the potency of preclinically tested agents and to better estimate the likelihood of their later clinical adoption into phase I-II trials. With this work, we provide an innovative model which reflects the BM microenvironment as a crucial predictor for in-vivo sensitivity as shown for ARRY-5200. This ex-vivo approach helps to better incorporate MM growth support by bone and BM-derived bystander cells and thus depicts a valid tool to better characterize the role of the BM niche in myeloma. Figure 1. Figure 1. Disclosures Engelhardt: Deutsch Krebshilfe: Other: grant. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

The Potential Oncogenic and MLN4924-Resistant Effects of CSN5 on Cervical Cancer Cells

2021 ◽  
Author(s):  
Huilin Zhang ◽  
Ping He ◽  
Qing Zhou ◽  
Yan Lu ◽  
Bingjian Lu
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Cell Proliferation ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Cervical Cancer Cell ◽  
Cervical Cancers ◽  
Cervical Cancer Cells

Abstract BackgroundsCSN5, a member of Cop9 signalosome, is essential for protein neddylation. It has been supposed to serve as an oncogene in some cancers. However, the role of CSN5 has not been investigated in cervical cancer yet.MethodsData from TCGA cohorts and GEO dataset was analyzed to examine the expression profile of CSN5 in cervical cancers. The role of CSN5 on cervical cancer cell proliferation was investigated in cervical cancer cell lines, Siha and Hela, through CSN5 knockdown via CRISPR-CAS9. Western blot was used to detect the effect of CSN5 knockdown and overexpression. CCK8, clone formation assay and cell cycle assay were also employed. Besides, the role CSN5 knockdown in vivo was evaluated by xenograft tumor model. Moreover, MLN4924 was applied in Siha and Hela with CSN5 overexpression.ResultsWe found that downregulation of CSN5 in Siha and Hela cells inhibited cell proliferation in vitro and in vivo, and the inhibitory effects were largely rescued by CSN5 overexpression. Moreover, deletion of CSN5 caused cell cycle arrest rather than inducing apoptosis. Importantly, CSN5 overexpression confers resistance to the anti-cancer effects of MLN4924 (pevonedistat) in cervical cancer cells.ConclusionsOur findings demonstrated that CSN5 functions as an oncogene in cervical cancers and may serve as a potential indicator for predicting the effects of MLN4924 treatment in the future.

scholarly journals Isolation of small, primitive human hematopoietic stem cells: distribution of cell surface cytokine receptors and growth in SCID-Hu mice

Blood ◽  
1995 ◽  
Vol 86 (2) ◽  
pp. 512-523 ◽  
Author(s):  
JE Wagner ◽  
D Collins ◽  
S Fuller ◽  
LR Schain ◽  
AE Berson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Surface ◽  
Cell Population ◽  
Size Fraction ◽  
Cell Surface Antigens ◽  
Inhibitory Protein ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cell Fractions

Human CD34+ cells were subfractionated into three size classes using counterflow centrifugal elutriation followed by immunoadsorption to polystyrene cell separation devices. The three CD34+ cell fractions (Fr), Fr 25/29, Fr 33/37, and Fr RO, had mean sizes of 8.5, 9.3 and 13.5 microns, respectively. The majority of cells in the large Fr RO CD34+ cell population expressed the committed stage antigens CD33, CD19, CD38, or HLA-DR and contained the majority of granulocyte- macrophage colony-forming units (CFU-GM), burst-forming units-erythroid (BFU-E), and CFU-mixed lineage (GEMM). In contrast, the small Fr 25/29 CD34+ cells were devoid of committed cell surface antigens and lacked colony-forming activity. When seeded to allogeneic stroma, Fr RO CD34+ cells produced few CFU-GM at week 5, whereas cells from the Fr 25/29 CD34+ cell population showed a 30- to 55-fold expansion of myeloid progenitors at this same time point. Furthermore, CD34+ cells from each size fraction supported ontogeny of T cells in human thymus/liver grafts in severe combined immunodeficient (SCID) mice. Upon cell cycle analyses, greater than 97% of the Fr 25/29 CD34+ cells were in G0/G1 phase, whereas greater proportions of the two larger CD34+ cell fractions were in active cell cycle. Binding of the cytokines interleukin (IL)-1 alpha, IL-3, IL-6, stem cell factor (SCF), macrophage inhibitory protein (MIP)-1 alpha, granulocyte colony- stimulating factor (G-CSF), and granulocyte-macrophage (GM)-CSF to these CD34+ cell populations was also analyzed by flow cytometry. As compared with the larger CD34+ cell fractions, cells in the small Fr 25/29 CD34+ cell population possessed the highest numbers of receptors for SCF, MIP1 alpha, and IL-1 alpha. Collectively, these results indicate that the Fr 25/29 CD34+ cell is a very primitive, quiescent progenitor cell population possessing a high number of receptors for SCF and MIP1 alpha and capable of yielding both myeloid and lymphoid lineages when placed in appropriate in vitro or in vivo culture conditions.

scholarly journals Interplay between PI3K/mTOR Signaling and IRE1a-XBP1 Promotes Survival of Pre-B NRASG12D all Cells Providing a Therapeutic Vulnerability for the "Undruggable" Driver RAS

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 47-48
Author(s):  
Iris Appelmann ◽  
Azam Salimi ◽  
Michael Huber ◽  
Mirle Schemionek ◽  
Margherita Vieri ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Signaling Pathway ◽  
Research Funding ◽  
Mtor Signaling ◽  
Ras Signaling ◽  
Genetic Ablation ◽  
Downstream Targets ◽  
Pharmacological Inhibition ◽  
Viable Cells

Background Activating RAS mutations drive around 30% of pre-B cell acute lymphoblastic leukemias (pre-B ALL) and are particularly common in relapsed ALL with a consecutively poor outcome. Recently published data demonstrated the critical role of the unfolded protein response (UPR) network, namely its IRE1α-XBP1 axis, for the survival of pre-B ALL cells: High expression of XBP1 confers a poor prognosis in pre B-ALL. However, the mechanism of XBP1 activation has not yet been elucidated in RAS mutated pre-B ALL. In this study, we aimed at identifying the molecular mechanism underlying pro-survival IRE1α-XBP1 signaling in RAS mutated pre-B ALL. Methods For a TET-ON inducible NRASG12D model in conditional Xbp1 knockout mice, we used interleukin 7 (IL-7)-dependent murine Mx1-Cre;Xbp1fl/fl pre-B cells transduced with a TET-ON inducible NRASG12D. We performed in vitro cell cycle and apoptosis assays with propidium iodide (PI) and annexin-V/PI. Furthermore, Western Blot and RT-qPCR were applied to analyze target gene expression. In a second approach, we focused on the signaling events following the blockade of RAS downstream targets using the MEK inhibitor PD0325901 and the dual PI3K/mTOR inhibitor BEZ235. We then assessed the efficacy of small molecule inhibition of IRE1α by MKC-8866 on XBP1 inactivation in RAS-mutated pre-B ALL cells either as a single treatment and in combination with the above mentioned drugs. Results We found the expression of Xbp1 significantly increased at the mRNA level with induction of NRASG12D. To determine the significance of Xbp1 in NRASG12D-driven pre-B ALL, we genetically deleted the IRE1α target Xbp1 using Cre-mediated deletion of Xbp1fl/fl in our mouse model of pre-B ALL. Genetic loss of Xbp1 significantly induced apoptosis (2.0-fold, p&lt;0.0001) and caused cell cycle arrest (induction of G0/1, 1.7-fold, p=0.0003) along with an increase in the expression of CDK inhibitors, p21CIP1 and p27KIP1 at the protein level. Genetic ablation of Xbp1 abrogated IL-7 receptor (IL-7R) signaling by reducing the phosphorylation levels of STAT5-Y694 and JAK1-Y1022/Y1023. In an additional approach, we revealed that IL-7-deprived pre-B ALL cells reduce the mRNA expression of Xbp1s, indicating that Xbp1 acts as a downstream linchpin of the IL-7 receptor signaling pathway. Both IL-7-deprivation and genetic loss of Xbp1 increased the phosphorylation levels of ERK1/2-T202/Y204, AKT-S473 and the protein levels of NRASG12D and MAPK negative regulator DUSP6. Pharmacological inhibition of XBP1 activation using MKC-8866 resulted in similar effects on the expression of RAS downstream targets. We therefore tested MKC-8866 in combination with MEK inhibition by PD0325901 as a potential therapeutic strategy against pre-B ALL, which proved non-efficient. As a second option with therapeutic implications, we focused on the PI3K pathway which acts downstream of both the IL-7R and RAS signaling pathways. Strikingly, we observed that genetic ablation of Xbp1 (viable cells after 72 h, BEZ: 71.9 ± 9.0 vs BEZ+ Mx1-Cre;Xbp1fl/f: 10.0 ± 4.9) or pharmacological inhibition of its production with MKC-8866 (viable cells after five days, BEZ: 58.0 ± 6.8 vs BEZ+ MKC-8866: 13.3 ± 7.4) sensitizes pre-B ALL to dual inhibition of PI3K/mTOR with BEZ235. By applying the Bliss formula, we were able to show that BEZ235 in combination with MKC-8866 synergistically reduces the viability of RAS-mutated pre-B ALL cells. Gene expression analysis indicated that BEZ235 in combination with MKC-8866 fully blocked IL-7R signaling and caused an aberrant activation of Ras-Erk signaling. Targeting PI3K/mTOR signaling along with XBP1 inactivation increased expression of NRASG12D and its target DUSP6. In addition, we showed that combined therapy increased expression levels of p19Arf in RAS-mutated pre-B ALL, implicating cell senescence mediated by activated RAS signaling. Conclusion Our work strongly supports the hypothesis that XBP1 induces its positive effects on progression of pre-B ALL cells through the IL-7R signaling pathway. IL-7R signaling through its downstream effector XBP1 counteracts the RAS signaling pathway to promote leukemogenesis in pre-B ALL cells. Active XBP1 prevents the cytotoxic effects of BEZ235 in pre-B ALL cells, and hence targeting XBP1 in combination with dual PI3K/mTOR inhibition by BEZ235 appears as a promising targeted strategy against the "undruggable" driver RAS in NRASG12D-mutated pre-B ALL. Disclosures Brümmendorf: Janssen: Consultancy; Merck: Consultancy; Novartis: Consultancy, Other: travel, accommodation, expenses, Research Funding; Takeda: Consultancy; Pfizer: Consultancy, Honoraria, Other: Travel, Accommodation, Expenses, Research Funding.

scholarly journals Phenotype Does Not Always Equal Function: HDAC Inhibitors and UM171, but Not SR1, Lead to Rapid Upregulation of CD90 on Non-Engrafting CD34+CD90-Negative Human Cells

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 659-659
Author(s):  
Kevin A. Goncalves ◽  
Megan D. Hoban ◽  
Jennifer L. Proctor ◽  
Hillary L. Adams ◽  
Sharon L. Hyzy ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Small Molecule ◽  
Hdac Inhibitors ◽  
Employment Equity ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Equity Ownership ◽  
Human Hscs

Abstract Background. The ability to expand human hematopoietic stem cells (HSCs) has the potential to improve outcomes in HSC transplantation and increase the dose of gene-modified HSCs. While many approaches have been reported to expand HSCs, a direct comparison of the various methods to expand transplantable HSCs has not been published and clinical outcome data for the various methods is incomplete. In the present study, we compared several small molecule approaches reported to expand human HSCs including HDAC inhibitors, the aryl hydrocarbon antagonist, SR1, and UM171, a small molecule with unknown mechanism, for the ability to expand phenotypic HSC during in vitro culture and to expand cells that engraft NSG mice. Although all strategies increased the number of phenotypic HSC (CD34+CD90+CD45RA-) in vitro, SR1 was the most effective method to increase the number of NOD-SCID engrafting cells. Importantly, we found that HDAC inhibitors and UM171 upregulated phenotypic stem cell markers on downstream progenitors, suggesting that these compounds do not expand true HSCs. Methods. Small-molecules, SR1, HDAC inhibitors (BG45, CAY10398, CAY10433, CAY10603, Entinostat, HC Toxin, LMK235, PCI-34051, Pyroxamide, Romidepsin, SAHA, Scriptaid, TMP269, Trichostatin A, or Valproic Acid) and UM171 were titrated and then evaluated at their optimal concentrations in the presence of cytokines (TPO, SCF, FLT3L, and IL6) for the ability to expand human mobilized peripheral blood (mPB)-derived CD34+ cells ex vivo . Immunophenotype and cell numbers were assessed by flow cytometry following a 7-day expansion assay in 10-point dose-response (10 µM to 0.5 nM). HSC function was evaluated by enumeration of colony forming units in methylcellulose and a subset of the compounds were evaluated by transplanting expanded cells into sub-lethally irradiated NSG mice to assess engraftment potential in vivo . All cells expanded with compounds were compared to uncultured or vehicle-cultured cells. Results. Following 7 days of expansion, SR1 (5-fold), UM171 (4-fold), or HDAC inhibitors (&gt;3-35-fold) resulted in an increase in CD34+CD90+CD45RA- number relative to cells cultured with cytokines alone; however, only SR1 (18-fold) and UM171 (8-fold) demonstrated enhanced engraftment in NSG mice. Interestingly, while HDAC inhibitors and UM171 gave the most robust increase in the number and frequency of CD34+CD90+CD45RA- cells during in vitro culture, these methods were inferior to SR1 at increasing NSG engrafting cells. The increase in CD34+CD90+CD45RA- cells observed during in vitro culture suggested that these compounds may be generating a false phenotype by upregulating CD90 and down-regulating CD45RA on progenitors that were originally CD34+CD90-CD45RA+. We tested this hypothesis by sorting CD34+CD90-CD45RA+ cells and culturing these with the various compounds. These experiments confirmed that both HDAC inhibitors (33-100 fold) and UM171 (28-fold) led to upregulation of CD90 on CD34+CD90-CD45RA+ cells after 4 days in culture. Since approximately 90% of the starting CD34+ cells were CD90-, these data suggest that most of the CD34+CD90+CD45RA- cells in cultures with HDAC inhibitors and UM171 arise from upregulation of CD90 rather than expansion of true CD34+CD90+CD45RA- cells and may explain the disconnect between in vitro HSC phenotype and NSG engraftment in vivo . This was further confirmed by evaluation of colony forming unit frequency of CD34+CD90-CD45RA+ cells after culture with compounds. Conclusions. We have showed that AHR antagonism is optimal for expanding functional human HSCs using the NSG engraftment model. We also demonstrated that UM171 and HDAC inhibitors upregulate phenotypic HSC markers on downstream progenitors. This could explain the discrepancy between impressive in vitro phenotypic expansion and insufficient functional activity in the NSG mouse model. Therefore, these data suggest caution when interpreting in vitro expansion phenotypes without confirmatory functional transplantation data, especially as these approaches move into clinical trials in patients. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership. Hoban: Magenta Therapeutics: Employment, Equity Ownership. Proctor: Magenta Therapeutics: Employment, Equity Ownership. Adams: Magenta Therapeutics: Employment, Equity Ownership. Hyzy: Magenta Therapeutics: Employment, Equity Ownership. Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

scholarly journals Preclinical Activity of the MPS1 Inhibitor S81694 in Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2631-2631
Author(s):  
Anna Kaci ◽  
Emilie Adiceam ◽  
Melanie Dupont ◽  
Marine Garrido ◽  
Jeannig Berrou ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Solid Tumors ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Jurkat Cells ◽  
Cell Cycle Distribution ◽  
Monopolar Spindle

Introduction: The dual-specificity protein kinase, monopolar spindle 1 (Mps1) is one the main kinases of the spindle assembly checkpoint (SAC) critical for accurate segregation of sister chromatids during mitosis. A hallmark of cancer cells is chromosomal instability caused by deregulated cell cycle checkpoints and SAC dysfunction. Mps1 is known to be overexpressed in several solid tumors including triple negative breast cancer. Thus, Mps1 seems to be a promising target and small molecules targeting Mps1 entered clinical trials in solid tumors. ALL originates from malignant transformation of B-and T-lineage lymphoid precursors with a variety of genetic aberrations including chromosome translocations, mutations, and aneuploidies in genes responsible for cell cycle regulation and lymphoid cell development. While outcome is excellent for pediatric patients and younger adults, relapsed and refractory disease still remain a clinical challenge for elder patients. Here, we demonstrate for the first time preclinical efficacy of the small molecule Mps1 inhibitor (Mps1i) S81694 in T- and B- ALL cells including BCR-ABL1+-driven B-ALL. Materials and Methods: Expression of Mps1 was determined by RT-qPCR and WB in JURKAT, RS4-11 and BCR-ABL1+ cells (BV-173 and TOM-1). A small molecule Mps1i (S81694) was tested alone (0 to 1000nM) or in combination with imatinib, dasatinib, nilotinib and ponatinib in BCR-ABL1+ ALL cell lines. Cell viability and IC50 was assessed by MTS assays after exposure to Mps1i for 72h. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTS assays and combination index (CI) values were calculated according to the Bliss model. Induction of apoptosis was evaluated by annexin-V exposure and PI incorporation at 72h with increasing doses of Mps1i. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) intercalation at 48h. Phosphorylation of Mps1 was detected in synchronized (by nocodazole and MG-132) cells by immunofluorescence using an anti phospho-Mps1 antibody detecting Thr33/Ser37 residues. Time-lapse microscopy was used in cell lines in presence or absence of S81694 to determine mitosis duration. Bone marrow (BM) nucleated patient cells were obtained after informed consent and incubated in methylcellulose with cytokines with or without Mps1i for 2 weeks to determine colony growth. Results: Expression of Mps1 could be detected by RT-qPCR and at the protein level by WB in all cell lines (Figure 1A and B ). IC50 after Mps1i exposure alone was 126nM in JURKAT cells, 51nM in RS4-11 cells, 75nM in BV-173 cells and 83nM in TOM-1. Significant apoptosis as detected by phosphatidylserine exposure and PI incorporation in all cell lines with BCR-ABL1+ cell lines BV-173 and TOM-1 cells being the most sensitive (80% and 60% apoptotic cells respectively)(Figure 1C). Upon Mps1i exposure we observed targeted inhibition of Mps1 phosphorylation at Thr33/Ser37 residues indicating the specific on target effect of S81694 by inhibiting Mps1 autophosphorylation (Figure 1D and E). Cell cycle profile was generally lost after treatment with S81694 in all cell lines indicating aberrant 2n/4n distribution due to SAC abrogation (Figure 1F). Furthermore, we demonstrated that S81694 exposure accelerated significantly mitosis in BV-173 cell line from 36 minutes to 19 minutes indicating effective inhibition of SAC function (Figure 1G). Interestingly, S81694 induced significant apoptosis (70%) in the imatinib resistant BV173 cell line bearing the E255K-BCR-ABL1-mutation. Combination of S81694 with TKI imatinib, dasatinib and nilotinib (but not ponatinib) was strongly synergistic in BCR-ABL1+ cells (Figure 1H). Finally, we observed inhibition of colony formation in a patient with BCR-ABL1+ B-ALL after exposure to 100nM and 250nM S81694 (reduction of 85% and 100% respectively)(Figure 1I). Conclusion: Mps1i S81694 yields significant preclinical activity in T-and B-cell ALL including BCR-ABL1+ models. Interestingly S81694 was efficacious in a TKI resistant cell line. Disclosures Kaci: Institut de Recherches Internationales Servier (IRIS): Employment. Garrido:Institut de Recherches Internationales Servier (IRIS): Employment. Burbridge:Institut de Recherches Internationales Servier (IRIS): Employment. Dombret:AGIOS: Honoraria; CELGENE: Consultancy, Honoraria; Institut de Recherches Internationales Servier (IRIS): Research Funding. Braun:Institut de Recherches Internationales Servier (IRIS): Research Funding.

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