scholarly journals Production of TGF-β Is Decreased in the Bone Marrow of Double Knockout (DKO) SMAD3-/- Fancd2-/- Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4798-4798
Author(s):  
Virginia Falvello ◽  
Michael W. Epperly ◽  
Tracy Dixon ◽  
Darcy Franicola ◽  
Xichen Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Mouse Strain ◽  
Colony Formation ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Double Knockout ◽  
Mouse Tissues ◽  
Organ Specific

Abstract Homologous deletion recombinant negative DKO mice, missing action of both Fancd2 and SMAD3 proteins were derived to test the hypothesis that abrogation of TGF-β signaling would reverse Fanconi Anemia (FA) stem cell sensitivity to TGF-β. To determine whether DKO mouse tissues demonstrated detectable differences in production of the ligand for TGF-β receptor, tissues from adult six - eight week old DKO mice were compared to those from Fancd2-/- (C57BL/6J background), SMAD3-/- (129/Sv genetic background), or wild type F1 mice (129/Sv X B6) for levels of TGF-β. Tissues examined included bone marrow, intestine, spleen, liver, muscle, and brain. Tissues were homogenized, and analyzed for bone marrow protein by Luminex analysis using a TGF beta 1 multispecies kit for Luminex Platform (Life Technologies). The results demonstrated decreased but not significant production of TGF-β in the bone marrow of DKO mice (3068 ± 753 pg TGF-β/mg protein) compared to 5211 ± 1662 (p = 0.0761) for wild type F1 mice (129/Sv X B6), and 5192 ± 705 (0.1085) for Fancd2-/- mice. However, TGF-β production in DKO was significantly decreased compared to Smad3-/- (129/Sv) mice (9828 ± 1076, p = 0.0127). The lungs from DKO also had decreased TGF-β production compared to wild type F1 mice (436 ± 125 and 1159 ± 44 pg/mg, respectively, p= 0.0217). Decreased TGF-β production was also seen in the DKO liver compared to F1 wild type liver (13.2 ± 1.7 and 33.5 ± 3.6, respectively, p = 0.0072). Bone marrow stromal cell lines derived from long-term bone marrow cultures of each mouse strain were tested for production of TGF-β. SMAD3-/- bone marrow stromal cells also had an increased production of TGF-β (236 pg/mg) compared to wild type F1, Fancd2-/- and DKO cell lines (117, 136 and 144 pg/ml). Bone marrow CFU-GEMM from each mouse strain was tested for sensitivity to inhibition by increasing concentrations of TGF-β, and both fresh bone marrow from DKO and SMAD3-/- mice demonstrated no TGF-β mediated abrogation of colony formation. In contrast, fresh marrow from wild type and Fancd2-/- mice demonstrated TGF-β concentration dependent inhibition of CFU-GEMM colony formation in vitro. These data indicate that TGF-β production in DKO mice is decreased in bone marrow, lung and liver compared to that in F1 wild type, SMAD3-/- or Fancd2-/- mice, and suggest that the control of TGF-β production by abrogation of TGF-β signaling in the setting of deletion of Fancd2 may be modulated in an organ specific manner. Supported by research grant NIAID/NIH, U19A168021. Disclosures No relevant conflicts of interest to declare.

Abstract 470: Recording Cell Migration Dynamics in Mouse Tissues With Cells Secreting Fluorescence Protein-tagged Collagen

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Zhongming Chen
Keyword(s):  
Cell Migration ◽  
Cell Lines ◽  
Wild Type Mouse ◽  
Cardiac Progenitor Cells ◽  
Wild Type ◽  
Mouse Tissues ◽  
Migration Dynamics ◽  
Fluorescence Protein

Background: Cell migration is an important step involved in heart regeneration and many cardiovascular diseases. However, cell migration dynamics in vivo is poorly understood due to the challenges from mammal hearts, which are opaque and fast beating, and thus individual cardiac cells cannot be imaged or tracked. Aims: In this study, cell migration dynamics in the heart is recorded with a novel strategy, in which fluorescence protein-tagged collagen is secreted from cells and deposited into extracellular matrix, forming visible trails when cells are moving in tissues. As a proof-of-concept, transplanted migration dynamics of cardiac progenitor cells in mouse hearts were investaged. Methods: Stable cell lines expressing mCherry-tagged type I collagen were generated from isolated cardiac progenitor cells, ABCG2 + CD45 - CD31 - cells (side populations), or c-kit + CD45 - CD31 - cells (c-kit + CPCs). The cell migration dynamics were monitored and measured based on the cell trails after cell transplantation into mouse tissues. Results: The stable cell lines form red cell trails both in vitro and in vivo (Fig. 1A & 1B, Green: GFP; Red: mCherry-collagen I, Blue: DAPI, bar: 50 microns). In culture dishes, the cells form visible cell trails of fluorescence protein. The cell moving directions are random, with a speed of 288 +/- 79 microns/day (side populations, n=3) or 143 +/-37 microns/day (c-kit + CPCs, n=3). After transplantation into wild-type mouse hearts, the cells form highly tortuous trails along the gaps between the heart muscle fibers. Angle between a cell trail and a muscle fiber is 16+/-16 degree (n=3). Side populations migrate twice as fast as c-kit+ CPCs in the heart (16.0 +/-8.7 microns/day vs. 8.1+/-0.0 microns/day, n=3, respectively), 18 time slower than the respective speeds in vitro . Additionally, side populations migrate significantly faster in the heart than in the skeletal muscles (26.4+/-5.8 microns/day, n=3). The side populations move significantly faster in immunodeficient mouse hearts (36.7+/-13.3 microns/day, n=3, typically used for studying cell therapies) than in wild-type mouse hearts. Conclusion: For the first time, cell migration dynamics in living hearts is monitored and examined with genetically modified cell lines. This study may greatly advance the fields of cardiovascular biology.

CD74 Expression by AML Cell Lines and Bone Marrow Specimens, and Augmented In Vitro Cytotoxicity of Anti-CD74 Antibody After Interferon-Gamma (IFN-γ) Treatment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2888-2888
Author(s):  
Abhinav B. Chandra ◽  
Jack Burton ◽  
Rhona Stein ◽  
Susan Chen ◽  
Nidhi Mishra ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cellular Signaling ◽  
In Vitro Cytotoxicity ◽  
Cancer Type ◽  
Phosphorylated Akt ◽  
Wide Range ◽  
Ifn Γ

Abstract Abstract 2888 Background: CD74 (HLA-DR-associated invariant chain) is expressed alone or along with DR in a wide range of hematologic cancers and solid tumors. Humanized anti-CD74 mAb, milatuzumab (Immunomedics, Morris Plains, NJ), exhibits direct cytotoxicity for NHL, CLL and MM cell lines, and is undergoing clinical evaluation for treatment of these malignancies. CD74 is upregulated by interferons in hematologic and epithelial cancer cell lines. Here we present the results of our analysis of CD74 expression and function in AML, and the effect of CD74 upregulation by treatment with IFN-γ on the cytotoxicity of milatuzumab for AML cell lines. Methods: CD74 expression in bone marrow biopsy (BMB) specimens from non-M3 AML patients was evaluated by immunohistochemistry and, for the 3 human AML cell lines, by flow cytometry, with/without permeabilization and with/without IFN-γ (40 and 200 U/mL). These cell lines were also tested in proliferation assays for responses to milatuzumab, with/without IFN-γ. Also, assessment of apoptosis and cellular signaling was performed. Results: In the initial group of AML cases, 13/14 BMB specimens showed moderate to strong CD74 expression by leukemic blasts, which was mostly intracellular, usually with a perinuclear distribution. Three AML cell lines also showed moderate to strong expression of CD74, which was mostly intracellular. Without IFN-γ, surface expression of CD74 was present, but IFN-γ treatment of these 3 lines resulted in upregulation of surface CD74 by 69–117%. Much higher levels of intracellular CD74 were observed in all 3 lines (with and without IFN-γ), with IFN-γ-induced upregulation of intracellular CD74 in all 3 lines (from 85%-868%; P<0.001). In 2/3 lines, IFN-γ increased milatuzumab-mediated growth inhibition (23.7 to 44.8% and -3.9 to 30.9%, P=0.01 and P<0.05, respectively). Cytotoxicity was in part due to apoptosis, as significant increases in Annexin V binding (P=0.01) were observed after treatment with IFN-γ plus milatuzumab. Initial experiments addressing cellular signaling suggest a role for AKT, because phosphorylated AKT levels increased (P=0.06) in response to IFN-γ + milatuzumab. Conclusions: CD74 is expressed in AML patient specimens and in AML cell lines, with the majority of CD74 expression found intracellularly. Cell surface and cytoplasmic expression of CD74 were upregulated in AML lines after IFN-γ exposure. This increased expression resulted in increased cytotoxicity of the anti-CD74 mAb, milatuzumab, in 2/3 AML lines. This effect was through apoptosis and involved the AKT pathway. Thus, AML is another cancer type where combined IFN-γ and milatuzumab treatment may be useful. Supported in part by NIH grant PO1-CA103985 (DMG). Disclosures: No relevant conflicts of interest to declare.

Regulation of Gata1 Expression by HS+3.5

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3864-3864
Author(s):  
Julia E Draper ◽  
William G Wood ◽  
Catherine Porcher ◽  
Paresh Vyas
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Transgene Expression ◽  
Conflicts Of Interest ◽  
Es Cells ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Haematopoietic Stem Cell ◽  
Wild Type ◽  
Foetal Liver

Abstract Abstract 3864 Precise regulation of Gata1 expression is required in order to control the balance between lymphoid/granulomonocytic (GM) and megakaryocytic-erythroid (MegE) specification, as well as to ensure correct differentiation of the MegE lineages. Transcriptional control is conferred in part by cis regulatory elements. An upstream enhancer, HS-3.5, and the erythroid first exon IE of Gata1 are necessary and sufficient to direct transgene expression in primitive but not definitive erythroid cells. Transgene expression in definitive red blood cells is restored by inclusion of an intronic DNaseI hypersensitive site, HS+3.5. Here we report the characterization of the HS+3.5 null embryonic stem cells and the HS+3.5 knockout mouse. In vitro differentiation of HS+3.5 null ES cells resulted in reduced myeloid and megakaryocytic colony formation compared to wild type. The ΔHS+3.5 ES cells retained normal primitive erythroid colony formation. ΔHS+3.5 definitive erythroid colony progenitors displayed a decreased sensitivity to Interleukin 3 (IL3) signalling compared to wild type. ΔHS+3.5 mice were viable and had normal blood counts and films. GM and erythroid progenitors also developed normally. However, there was a mild expansion of the E14.5 foetal liver Megakaryocytic Progenitor (MkP) compartment and an increase in Gata2 expression in both the bone marrow and foetal liver MkPs. Turning to Gata1, a decrease in Gata1 expression was observed in the following compartments: the bone marrow long term haematopoietic stem cell (LT-HSC) and the foetal liver common myeloid progenitor (CMP). The relationship between the effect of the HS+3.5 deletion on Gata1 expression and the haematopoietic phenotype will be discussed. Disclosures: No relevant conflicts of interest to declare.

Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 859-859 ◽  
Author(s):  
Chen Zhao ◽  
Yan Xiu ◽  
John M Ashton ◽  
Lianping Xing ◽  
Yoshikazu Morita ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Double Knockout ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Physiological Processes ◽  
Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals IL-6 Deficiency Reverses Leukemic Transformation in an MDS Mouse Model

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 36-36
Author(s):  
Yang Mei ◽  
Yijie Liu ◽  
Xu Han ◽  
Jing Yang ◽  
Peng Ji
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Inflammatory Cytokines ◽  
Conflicts Of Interest ◽  
Double Knockout ◽  
Leukemic Transformation ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Age Related

Myelodysplastic syndromes (MDS) are a group of age-related myeloid malignancies that are characterized by ineffective hematopoiesis and increased incidence of developing acute myeloid leukemia (AML). The mechanisms of MDS to AML transformation are poorly understood, which is partially due to the scarcity of leukemia transformation mouse models. Recently, we established a mDia1/miR146a double knockout (DKO) mouse model mimicking human del(5q) MDS. DKO mice present with pancytopenia with aging due to myeloid suppressive cell (MDSC) expansion and over-secretion of pro-inflammatory cytokines including TNF-a and interlukine-6 (IL-6). In the current study, we found that most of the DKO mice underwent leukemic transformation at 12-14 months of age. The bone marrow of these mice was largely replaced by c-Kit+ blasts in a background of fibrosis. Flow cytometry analysis and in vitro colony formation assay demonstrated that hematopoietic stem progenitor cells (HSPCs) in DKO bone marrow were dramatically declined. The leukemic DKO mice had elevated white blood cell counts and circulating blasts, which contributes to the myeloid cell infiltration in non-hematopoietic organs including liver and lung. Moreover, the splenocytes from DKO old mice efficiently reconstitute the hematopoiesis, but led to a 100% disease occurrence with rapid lethality in gramma irradiated recipient mice, suggesting the leukemic stem cells enriched in DKO spleen were transplantable. Given the significant roles of the inflammatory cytokines in the pathogenesis of the DKO mice, we crossed DKO mice with IL-6 knockout mice and generated mDia1/miR-146a/IL-6 triple knockout (TKO) mice. Strikingly, the TKO mice showed dramatic rescue of the leukemic transformation of the DKO mice in that all the aforementioned leukemic phenotypes were abolished. In addition, IL-6 deficiency normalized the cell comparts and prevented leukemic transplantation ability in TKO spleen. Single cell RNA sequencing analyses indicated that DKO leukemic mice had increased monocytic blast population with upregulation of Fn1, Csf1r, and Lgals1, that was completely diminished with IL-6 knockout. Through a multiplex ELISA, we found IL-6 deficiency attenuated the levels of multiple inflammatory cytokines in TKO serum. In summary, we report a mouse model with MDS leukemic transformation during aging, which could be reverted with the depletion of IL-6. Our data indicate that IL-6 could be a potential target in high risk MDS. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ap2a2 Is Crucial for LT-HSC Quiescence

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2642-2642
Author(s):  
Stephen B Ting ◽  
Sara Rhost ◽  
Sarah Ghotb
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Fold Increase ◽  
Wild Type ◽  
Lacz Reporter ◽  
Adult Survivor ◽  
Self Renewal ◽  
Post Transplant

Abstract Despite the relative rarity of haematopoietic stem cells (HSCs) within the blood system, functional heterogeneity is paramount to their ability to sustain lifelong blood production. The quiescent HSC sits at the functional apex possessed with self-renewal properties and the greatest repopulation output. We previously identified the gene, Ap2a2 as an enhancer of HSC function and its protein as a potential cell fate determinant in HSC asymmetric cell divisions (Ting SB et al., Blood 2012). Mechanistically, we hypothesise Ap2a2 induces a state of HSC quiescence. Using the Tet-On histone H2B-GFP mouse model (Foudi et al., Nat Biotech 2009), we have shown Ap2a2 to be highly and differentially expressed in the predominantly, G0 dormant CD150+48-LSK GFPhigh as opposed to the more cycling GFPlow HSC subpopulation. Competitive transplantation of Ap2a2- versus empty vector-transduced H2B-GFP HSCs results in a three-fold increase of the CD150+48-LSK GFPhigh HSC subpopulation. To further confirm the importance of Ap2a2 in haematopoiesis, we have constructed Ap2a2-LacZ reporter and constitutive Ap2a2 knockout (KO) mouse lines. The Ap2a2 LacZ reporter with b-galactosidase flow cytometry staining of bone marrow subpopulations confirmed high endogenous Ap2a2 expression in the CD150+48-LSK long-term (LT-) versus CD150-48-LSK short-term (ST-) repopulating HSCs. Interim analyses of the constitutive Ap2a2 KO mice have revealed two obvious phenotypes: 14% of Ap2a2-null mice termed "non-survivors" are smaller, paler with failure of fetal liver (FL) development and die between E18.5 and weaning, whilst the remaining 11% are adult viable "survivors". However, at E14.5, Ap2a2-null compared to Ap2a2-wild type fetal livers showed less absolute total FL cells but increased CD150+48-LSM FL HSCs. This was quantitatively correlated via limiting dilution assay assessed at 16 weeks post-transplant with a two-fold increase in Ap2a2-null HSC numbers (1 in 78,917 versus 1 in 150,891, p=0.027). This suggests Ap2a2 has a role in FL HSC differentiation and/or fate with potential impairment of symmetrical versus asymmetrical HSC divisions currently being studied. When E14.5 FL cells were competitively transplanted, the Ap2a2-null HSC had impaired donor reconstitution function measured at 16 weeks post-transplant (19.8% versus 48.6%, p=0.015). Ap2a2-null versus wild-type E14.5 FL cells showed equivalent numbers of primary in vitro methylcellulose colony assays but loss of secondary colonies upon re-plating indicative of loss of in-vitro HSC self-renewal. Importantly, although the Ap2a2 adult "survivors" exhibited normal quantities of bone marrow HSC subpopulations, when functionally assessed, Ap2a2-null adult "survivor" HSCs showed loss of in-vivo HSC self-renewal in secondary transplantation assays. To investigate potential cellular mechanisms, we studied the cell cycle state of Ap2a2-null and wild-type E14.5 FL cells and identified that Ap2a2-null "non-survivors" had a relative loss of quiescent G0, specifically in the LT-HSC (and not seen in the ST-HSC) subpopulation throughout all of (E14.5 to E18.5) FL development. In contrast, the LT-HSC subpopulation in FLs of Ap2a2-null "survivors" had an initial loss of G0 at E14.5 but a compensatory increase in LT-HSC G0 by E18.5. Our preliminary data suggests Ap2a2 is a crucial factor for the quiescent LT-HSC subpopulation, and we propose that both during the highly proliferative fetal liver stage of haematopoiesis and adult HSCs under stress that Ap2a2 maintains a critical balance of dormant ("deep-sleeper") HSCs to ensure global HSC function. Disclosures No relevant conflicts of interest to declare.

scholarly journals Radiosensitivity of Fancd2-/- mouse Bone Marrow Stromal Cells Is Altered By Abrogation of TGF-β Signaling

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4796-4796
Author(s):  
Katherine Chen ◽  
Darcy Franicola ◽  
Donna Shields ◽  
Michael W. Epperly ◽  
Xichen Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Survival Curve ◽  
Radiation Sensitivity ◽  
Mouse Bone Marrow ◽  
Wild Type ◽  
Increased Risk ◽  
Significant Difference

Abstract Both marrow-transplanted and non-transplanted Fanconi Anemia (FA) patients are often radiosensitive. Due to an increased risk of developing secondary malignancies, these patients require dose and volume modification during radiotherapy. To determine whether abrogation of TGF-β signaling alters the radiation sensitivity of Fancd2-/- mice, cell lines derived from double knockout (DKO) (SMAD3-/- Fancd2-/-) mice were compared with those from Fancd2-/-, SMAD3-/-, and wild-type mice for ionizing irradiation sensitivity. Bone marrow stromal cell lines were derived from long-term bone marrow cultures of DKO, Fancd2-/-, SMAD3-/-, and wild-type SMAD3+/+ (129/Sv) X Fancd2+/+ (B6) F1 mice. Radiation sensitivity was determined using clonogenic irradiation survival curves. There was no significant difference in radiosensitivity comparing DKO cells (Do = 1.95 ± 0.06 Gy, ň = 4.3 ± 0.7) to the wild type SMAD3+/+ (129/Sv) X Fancd2+/+ (B6) F1 cell line (Do = 2.00 ± 0.11 Gy, and ň = 5.1 ± 0.7, p = 0.7003 and 0.4820, respectively). The Fancd2-/- cell line was more radiosensitive with a Do of 1.37 ± 0.09 Gy compared to 1.95 ± 0.07 and 2.00 ± 0.11 for DKO and wild type cells (p = 0.0063 and 0.0360, respectively. In contrast, the SMAD3-/- cell line was more radioresistant with an increased shoulder on the irradiation survival curve (ň = 12.1 ± 2.9) compared to the DKO or wild type SMAD3+/+ (129/Sv) X Fancd2+/+ (B6) F1 cell lines (ň = 4.335 ± 0.7 or 5.1 ± 0.7, p = 0.00277 or 0.0426, respectively). This confirms and extends results with SMAD3-/- mouse derived cell lines on another background strain (C57BL/6J) (Epperly, et al., Radiation Research, 165:671-677, 2006). TGF-β signaling was abrogated in both DKO and SMAD3-/- mouse cell lines (measured by TGF-β inhibition of fresh marrow CFU-GEMM in vitro), confirming the phenotype of altered TGF-β signaling. Therefore, radiosensitivity associated with the Fancd2-/- genotype was abrogated by interruption of the TGF-β signaling pathway in the same cells. Supported by research grant NIAID/NIH, U19A168021. Disclosures No relevant conflicts of interest to declare.

Cytotoxic Activity of Anti-CD20-hIFN-α on Rituximab-Resistant B-NHL Clones and Synergy with Chemotherapy,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3499-3499 ◽  
Author(s):  
Gabriel G Vega ◽  
Mario I Vega ◽  
Sara Huerta-Yepez ◽  
Ali Jazirehi ◽  
Hector Mayani ◽  
...  
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Tumor Xenograft ◽  
Antibody Concentration ◽  
Wild Type ◽  
Anti Cd20 ◽  
Hodgkin's Lymphomas ◽  
Apoptotic Effects

Abstract Abstract 3499 Rituximab (chimeric anti-CD20 mAb) (Rtx) has been successfully used in the treatment of patients with B-cell non-Hodgkin's lymphomas (B-NHLs). The combination treatment with chemotherapy results in achieving high response rates and prolongation of survival. However, a subset of patients does not initially respond to treatment and many responding patients relapse and no longer respond to further treatments. Currently, there are no alternative therapies for resistant patients. The mechanism of resistance in vivo is not clear. However, we have explored a potential mechanism by developing in vitro several clones of Rtx-resistant (RR) variants for several B-NHL cell lines and characterized their properties. Briefly, unlike the parental wild-type, the RR clones express CD20 but no longer respond to treatment with Rtx or combination with cytotoxic drugs. Further, these clones overexpress the activity of several survival/anti-apoptotic pathways [1]. It is not known whether chemical modification of Rtx might be necessary to exert its activity and signaling on the RR clones. Hence, a recent report demonstrated that a fusion protein consisting of Rtx and human IFN-α (anti-CD20-hIFN-α) exhibited superior activity over Rtx, IFN-α, or combination of Rtx and IFN-α, and exhibited significant anti-proliferative and apoptotic effects in vitro with several B-NHL cell lines and in vivo an anti-tumor xenograft response [2]. These findings prompted us to investigate the effect of anti-CD20-hIFN-α on the RR clones. We hypothesized that anti-CD20-hIFN-α may exert an anti-proliferative and apoptotic effects on the RR clones and may also synergize when used in combination with chemotherapy. In this study, we used the B-NHL line Ramos (Burkitt) and 2F7 (AIDS-related) and their respective Ramos RR1 and 2F7 RR1 clones as models. We examined the effects of anti-CD20-hIFN-α and Rtx on the wild-type and RR clones following treatment with IgG isotype control, Rtx, anti-CD20-hIFN-α, CDDP (10 mg/ml) and Treanda® (Bendamustine) (5 mg/ml), as well as combinations. Treatment of 2F7 with single agents alone had no cytotoxic effect; however, treatment with the combination of Rtx and CDDP or Treanda® or anti-CD20-hIFN-α plus CDDP or Treanda® resulted in significant cytotoxicity. Treatment of Ramos resulted in similar findings observed with 2F7, however, the anti-CD20-hIFN-α alone was significantly cytotoxic to Ramos cells. Importantly, whereas treatment of 2F7 RR1 or Ramos RR1 with Rtx or Rtx plus CDDP or Treanda® had no cytotoxic effects (as expected), the treatment with the anti-CD20-hIFN-α alone had significant cytotoxicity and synergy was observed when used in combination with CDDP or Treanda®. In all of the above experiments, the level of cytotoxicity was a function of the antibody concentration used (range 10–30 μg/ml). The mechanism by which anti-CD20-hIFN-α signals the RR clones for cytotoxicity and sensitization was examined. Preliminary findings show that treatment of the RR clones with anti-CD20-hIFN-α inhibits the activity of p38MAPK survival pathway and also inhibits the anti-apoptotic gene products, Bcl-2/BclXL and upregulates the pro-apoptotic expression of Bax. These findings established, for the first time, that modification of Rtx by fusion with IFN-α was cytotoxic on the RR clones and synergized with chemotherapy. The findings also show, unlike Rtx that, anti-CD20-hIFN-α signals the RR cells and inhibits survival/antiapoptotic pathways leading to direct cytotoxicity and chemo-sensitization. The molecular signaling mediated by anti-CD20-hIFN-α on the cell membrane of RR cells leading to inhibition of survival pathways will be presented. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Development and characterization of prototypes for in vitro and in vivo mouse models of ibrutinib-resistant CLL

2021 ◽  
Vol 5 (16) ◽  
pp. 3134-3146
Author(s):  
Burcu Aslan ◽  
Gorkem Kismali ◽  
Lisa S. Chen ◽  
LaKesla R. Iles ◽  
Mikhila Mahendra ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Mouse Models ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Survival Duration ◽  
Wild Type ◽  
In Vivo Models

Abstract Although ibrutinib improves the overall survival of patients with chronic lymphocytic leukemia (CLL), some patients still develop resistance, most commonly through point mutations affecting cysteine residue 481 (C481) in Bruton’s tyrosine kinase (BTKC481S and BTKC481R). To enhance our understanding of the biological impact of these mutations, we established cell lines that overexpress wild-type or mutant BTK in in vitro and in vivo models that mimic ibrutinib-sensitive and -resistant CLL. MEC-1 cell lines stably overexpressing wild-type or mutant BTK were generated. All cell lines coexpressed GFP, were CD19+ and CD23+, and overexpressed BTK. Overexpression of wild-type or mutant BTK resulted in increased signaling, as evidenced by the induction of p-BTK, p-PLCγ2, and p-extracellular signal–related kinase (ERK) levels, the latter further augmented upon IgM stimulation. In all cell lines, cell cycle profiles and levels of BTK expression were similar, but the RNA sequencing and reverse-phase protein array results revealed that the molecular transcript and protein profiles were distinct. To mimic aggressive CLL, we created xenograft mouse models by transplanting the generated cell lines into Rag2−/−γc−/− mice. Spleens, livers, bone marrow, and peripheral blood were collected. All mice developed CLL-like disease with systemic involvement (engraftment efficiency, 100%). We observed splenomegaly, accumulation of leukemic cells in the spleen and liver, and macroscopically evident necrosis. CD19+ cells accumulated in the spleen, bone marrow, and peripheral blood. The overall survival duration was slightly lower in mice expressing mutant BTK. Our cell lines and murine models mimicking ibrutinib-resistant CLL will serve as powerful tools to test reversible BTK inhibitors and novel, non–BTK-targeted therapeutics.

Aurora A Kinase Is Required for Hematopoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1201-1201
Author(s):  
Benjamin Goldenson ◽  
Jeremy Q Wen ◽  
John Crispino
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Small Molecule ◽  
Dna Content ◽  
Colony Formation ◽  
Conflicts Of Interest ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Wild Type ◽  
Significant Difference

Abstract Abstract 1201 In acute megakaryocytic leukemia (AMKL), there is a failure of megakaryocytes to differentiate, become polyploid and stop dividing. We used an integrated screening approach that included chemical, proteomic and genetic screens to identify small molecules and their targets that control polyploidization and differentiation of normal and malignant megakaryocytes. We identified several small molecule inducers of polyploidy and used siRNA and proteomic target ID approaches to determine the cellular targets of the lead small molecule dimethylfasudil (diMF). Aurora Kinase A (AURKA) was identified as one of the top targets of diMF. AURKA is an attractive target in AMKL for several reasons. First, AURKA is overexpressed in AMKL cells. Second, at the biologically effective doses used in our cell-based assays, AURKA inhibition was selective for the megakaryocyte lineage. Third, AURKA inhibition by diMF or the selective AURKA inhibitor MLN8237 increased MK polyploidy, induced features of differentiation, blocked proliferation of AMKL blasts, and improved survival in an AMKL mouse model. AURKA is important in mitotic spindle assembly, mitosis, chromosomal alignment and segregation. Moreover, it is required for embryonic development, as Aurka−/− embryos fail to grow beyond the blastocyst stage. However, the extent to which AURKA is necessary for steady state hematopoiesis in adults is unknown. To investigate the necessity of AURKA in hematopoiesis, we utilized a conditionally targeted strain of mice (Aurkaflox/flox). To delete AURKA in megakaryocytes ex vivo, Aurkaflox/flox bone marrow cells were expanded, transduced with a retrovirus expressing Cre and GFP, and then cultured in the presence of THPO for 72 hours. We found that deletion of AURKA resulted in increased CD41 and CD42 expression as well as increased DNA content. Assays for apoptosis by Annexin V staining of Aurkaflox/flox cells infected with Cre also showed increased apoptosis in AURKA-deleted cells at 24 and 48 hours. To delete AURKA in vivo, we crossed Aurkaflox/flox mice to MX1-Cre mice and injected wild-type, heterozygous and homozygous floxed mice expressing MX1-Cre with pIpC every other day for six days. We found that deletion of AURKA in hematopoietic progenitors leads to pancytopenia, profound bone marrow defects and death within two weeks. Colony formation assays showed significantly decreased myeloid, erythroid and megakaryocyte colony formation with AURKA deficiency. Bone marrow histology displayed markedly hypocellular marrow, but curiously, flow cytometry revealed a significant increase in the percentage of CD41 and CD42 positive cells. This observation suggests that AURKA normally acts to restrain terminal differentiation of megakaryocytes and is consistent with the CD41 and CD42 inducing ability of AURKA inhibitors. To confirm that AURKA is the key target of our recently identified polyploidy inducers, we assayed the effects of diMF and MLN8237 on Aurka+/+, Aurka+/− and Aurka−/− megakaryocytes. 300 nM diMF and 100 nM MLN8237, concentrations that strongly induce polyploidy, did not increase MK polyploidization in Aurka−/− MKs. diMF and MLN8237 treatment increased polyploidy in Aurka+/− MKs with no significant difference in comparison to Aurka+/+ MKs. We also assayed the ability of wild-type or the T217D mutant of AURKA, which is resistant to inhibition by MLN8237, to reduce the induction of polyploidy caused by diMF and MLN8237 upon overexpression. CMK cells were infected with viruses harboring wild-type or T217D AURKA, treated with DMSO, 3 μM diMF or 30 nM MLN8237 for 72 hours, and then evaluated for DNA content. The increase in polyploidization induced by both compounds was significantly decreased in cells overexpressing the T217D mutant of AURKA. With overexpression of the wild-type AURKA, there was a trend towards reduction in polyploidy, but more variable effects and no significant difference. Thus, AURKA T217D overexpression reduced the ability of diMF and MLN8237 to induce polyploidization, consistent with our conclusion that diMF targets AURKA. Together, our data support a role of AURKA in megakaryocyte polyploidization and differentiation and show that AURKA is required for steady state hematopoiesis. The results also show that AURKA is the key target of diMF in the induction of polyploidization of megakaryocytes and support the development of Aurora A kinase inhibitors in clinical trials for AMKL. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document