scholarly journals Targeting Rac-Gtpases Reverses Stroma-Induced Resistance to Notch and mTOR-Inhibition in Acute T-Cell Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1326-1326
Author(s):  
Adrian Schwarzer ◽  
Martin May ◽  
Harald Genth ◽  
Zhixiong Li ◽  
Christopher Baum ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Notch Signaling ◽  
Induced Resistance ◽  
Stromal Cells ◽  
Cell Interactions ◽  
Mtor Inhibition ◽  
Rac Gtpases ◽  
Notch Inhibition ◽  
Cell Cell

Abstract Molecular hallmarks of T-ALL are the aberrant activation of NOTCH signaling and high activity of the PI3K-AKT-mTOR pathway. Upregulated mTOR and NOTCH have been linked to the resistance of T-ALL to chemotherapy as well as to high frequencies of leukemia-initiating cells. Hence, the mTOR and the NOTCH pathways are promising therapeutic targets in T-ALL. However, clinical success of the mTOR inhibitor Rapamycin in acute leukemia has been disappointing. Similar results have been observed in mouse models of T-ALL treated with Notch inhibitors. To investigate the impact of mTOR and Notch inhibition in a genetically complex T-ALL, we developed an aggressive murine T-ALL model, driven by tyrosine kinase signaling, loss of Pten, Cux1-haploinsufficiency and constitutive Notch signaling. In vitro, T-ALL blasts were highly sensitive to inhibition of AKT, mTOR and Notch signaling. We transplanted the leukemias into secondary recipients and initiated treatment with Rapamycin after the onset of leukemia. Rapamycin significantly prolonged survival of the animals (placebo: 27 days, Rapamycin 49 days, p<0.001). Eventually, all Rapamycin treated animals succumbed to the T-ALL that extensively infiltrated the bone marrow and solid organs despite continuous drug administration. When Rapamycin-resistant blasts were explanted and cultured in petri dishes they again became susceptible to Rapamycin, demonstrating a context-dependent resistance rather than outgrowth of intrinsically resistant clones. Gene set enrichment analysis revealed that Rapamycin-resistant T-ALL in vivo upregulated genetic networks associated with cell-cell interactions. Stromal cell support from OP9-cells as well as from mesenchymal stem cells recapitulated the in vivo effect and induced resistance to mTOR and Notch-inhibition in T-ALL blasts. Coating the tissue culture wells with Collagen, Fibronectin, Retronectin or Matrigel, did not elicit resistance. By using trans-well assays we show that the stroma-induced resistance was dependent on direct cell-cell interactions. Immunoblots and PhosFlow probing the mTORC1/C2 and Notch pathway demonstrated an identical drug effect on their intracellular targets in resistant T-ALL blasts cultured on stroma cells and susceptible cells in suspension. Since the number of molecules potentially involved in cell-to-cell contacts is vast, we focused on central nodes that organize this process in order to find a potentially druggable target that is critically involved in stroma-induced resistance. Transcriptome profiling pointed towards upregulation of Rac-associated pathways. We determined the activation of Rac1 by PAK-pull down assays in T-ALL blasts grown in suspension or on stromal cells. We observed an increase (FC=1.96 ± 0.58, p=0.04) in activated Rac1 in the T-ALL blasts in contact with a stromal layer. To determine whether Rac activation plays a role in stroma-induced resistance, we devised a strategy to abrogate Rac signaling in T-ALL blasts, but not in the stromal cells, since inhibition of Rac in stromal cells by the Rac-inhibitor NSC23766 led to the their detachment. Furthermore, Rac1,2 and 3 can be functionally redundant, making knock down experiments using shRNAs challenging. The Clostridium difficile serotype F strain 1470 produces toxin B isoform (TcdBF), that selectively glucosylates and inactivates Rac(1,2,3). We pretreated T-ALL blasts with TcdBF and observed a dose-dependent functional inhibition of Rac GTPases monitored by dephosphorylation of the Rac effector kinase pS144/141-PAK-1/2. T-ALL blasts were then incubated for 5 hours with increasing toxin doses, washed 3 times and incubated in toxin-free medium. Eighteen hours after the end of the exposure to the toxins, Rac was still inhibited. Strikingly, in the TcdBF-pretreated T-ALL, the stroma-induced resistance effect was abrogated and clusters of apoptotic cells were clearly visible (>2 fold reduction of the input, p=0.002). In contrast, the carrier-treated T-ALL exhibited resistance to the inhibitors on stroma (>10 fold expansion of the input, p<0.0003). Altogether, we identify the Rac-GTPases as a nexus of stroma-induced drug resistance and show that inhibition of Rac and mTOR is synthetically lethal to T-ALL blasts T-ALL blasts that are in contact with stromal cells, paving the way to augment the effectiveness of small molecule inhibitors in acute leukemia. Disclosures No relevant conflicts of interest to declare.

Runx1 Is Required for Notch-Induced Specification of Megakaryocyte Development from Early Hematopoietic Stem and Progenitor Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1370-1370
Author(s):  
Melanie G Cornejo ◽  
Thomas Mercher ◽  
Joseph D. Growney ◽  
Jonathan Jesneck ◽  
Ivan Maillard ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Cell Fate ◽  
Stromal Cells ◽  
Gfp Expression ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Insight Into ◽  
Megakaryocyte Differentiation

Abstract The Notch signaling pathway is involved in a broad spectrum of cell fate decisions during development, and in the hematopoietic system, it is known to favor T cell- vs B cell lineage commitment. However, its role in myeloid lineage development is less well understood. We have shown, using heterotypic co-cultures of murine primary hematopoietic stem cells (Lin-Sca-1+ckit+ HSCs) and OP9 stromal cells expressing the Notch ligand Delta1 (OP9-DL1), that Notch signaling derived from cell non-autonomous cues acts as a positive regulator of megakaryocyte fate from LSK cells. Bone marrow transplantation experiments with a constitutively active Notch mutant resulted in enhanced megakaryopoiesis in vivo, with increased MEP numbers and megakaryocyte colony formation. In contrast, expression of dnMAML using a conditional ROSA26 knock-in mouse model significantly impaired megakaryopoiesis in vivo, with a marked decrease in megakaryocyte progenitors. In order to understand the cellular differentiation pathways controlled by Notch, we first examined the ability of various purified progenitor populations to differentiate toward megakaryocytes upon Notch stimulation in vitro. We observed that CMP and MEP, but not GMP, can engage megakaryopoiesis upon Notch stimulation. Our results were consistent with expression analysis of Notch signaling genes in these purified progenitors and were supported by the observation that transgenic Notch reporter mice display higher levels of reporter (i.e. GFP) expression in HSC and MEP, vs. CMP and GMP in vivo. Furthermore, purified progenitors with high GFP expression gave rise to increased numbers of megakarocyte-containing colonies when plated in vitro compared to GFP-negative progenitors. In addition, further purification of the HSC population into long-term (LT), short-term (ST), and lymphoid-primed myeloid progenitors (LMPP) before plating on OP9-DL1 stroma showed that LMPP have a reduced ability to give rise to megakaryocytes compared to the other two populations. These data support the hypothesis that there is an early commitment to erythro/megakaryocytic fate from HSC prior to lymphoid commitment. To gain insight into the molecular mechanism underlying Notch-induced megakaryopoiesis, we performed global gene expression analysis that demonstrated the engagement of a megakaryopoietic transcriptional program when HSC were co-cultured with OP9-DL1 vs. OP9 stroma or OP9-DL1 treated with gamma-secretase inhibitor. Of interest, Runx1 was among the most upregulated genes in HSC co-cultured on OP9-DL1 stroma. To assess whether Notch signaling engages megakaryocytic fate through induction of Runx1, we plated HSC from Runx1 −/− mice on OP9-DL1 stroma. Compared to WT cells, Runx1 −/− HSC had a severely reduced ability to develop into CD41+ cells. In contrast, overexpression of Runx1 in WT HSC was sufficient to induce megakaryocyte fate on OP9 stroma without Notch stimulation. Together, our results indicate that Notch pathway activation induced by stromal cells is an important regulator of cell fate decisions in early progenitors. We show that Notch signaling is upstream of Runx1 during Notch-induced megakaryocyte differentiation and that Runx1 is an essential target of Notch signaling. We believe that these results provide important insight into the pathways controlling megakaryocyte differentiation, and may have important therapeutic potential for megakaryocyte lineage-related disorders.

In Vivo Blockade of Individual Notch Ligands and Receptors Provides a New Targeted Therapeutic Approach In Graft-Versus-Host Disease

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 819-819
Author(s):  
Ivy T Tran ◽  
Ashley R Sandy ◽  
Alexis Carulli ◽  
Gloria T Shan ◽  
Vedran Radojcic ◽  
...  
Keyword(s):  
T Cells ◽  
Notch Signaling ◽  
Gamma Secretase ◽  
Notch Receptors ◽  
Alloreactive T Cells ◽  
Donor T Cells ◽  
Notch Inhibition ◽  
Total Body ◽  
Notch Ligands

Abstract Abstract 819 Notch signaling is a cell-cell communication pathway with multiple functions in health and disease. Notch ligands of the Delta-like (Dll1, 3, 4) or Jagged (Jagged1, 2) family interact with one of four mammalian Notch receptors (Notch1-4), leading to proteolytic activation of the receptors by gamma-secretase. We have discovered a critical role for Notch signaling in the differentiation of pathogenic host-reactive T cells during graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT). Expression of the pan-Notch inhibitor DNMAML in donor T cells led to markedly reduced GVHD severity, without causing global immunosuppression (Blood 2011, 117(1): 299–308). These findings identify Notch signaling in alloreactive T cells as an attractive therapeutic target after allo-BMT. To explore preclinical strategies of Notch blockade in GVHD, we first assessed the effects of systemic pan-Notch inhibition with gamma-secretase inhibitors. In the B6 anti-BALB/c MHC-mismatched model of allo-BMT, administration of the gamma-secretase inhibitor dibenzazepine was as efficient as genetic strategies at blocking Notch target gene expression and production of inflammatory cytokines in donor T cells (IFN-γ, TNF-α, IL-2). However, dibenzazepine induced severe gastrointestinal toxicity after total body irradiation due to inhibition of both Notch1 and Notch2 in the gut epithelium. To avoid these side effects, we hypothesized that targeting individual Notch receptors or ligands could provide safe therapeutic Notch blockade after allo-BMT. Among the four mammalian Notch receptors (Notch1-4), donor alloreactive T cells expressed Notch1 and Notch2. Host dendritic cells expressed Notch ligands of the Jagged and Delta-like (Dll) families, with markedly increased Dll4 but not Jagged1/2 transcripts after total body irradiation. This suggested that blockade of Notch1 and/or Notch2 in T cells or Delta-like Notch ligands in dendritic cells could abrogate GVHD. To explore this possibility, we used specific monoclonal antibodies to neutralize Notch receptors and ligands in vivo after allo-BMT (Nature 2006, 444(7122):1083–7; Nature 2010, 464(7291): 1052–7). Combined blockade of Notch1 and Notch2 in vivo reduced the production of key inflammatory cytokines by alloreactive CD4+ and CD8+ T cells to a similar extent as DNMAML-mediated pan-Notch inhibition. Inhibition of Notch1 alone led to a large decrease in cytokine secretion, indicating that Notch1 is a dominant non-redundant Notch receptor in alloreactive T cells. Consistently, transplantation of Notch1-deficient but not Notch2-deficient B6 T cells allowed for decreased GVHD and improved survival in BALB/c recipients, similarly to global Notch inhibition by DNMAML. We then studied the consequences of inhibiting Dll1, Dll4 or both Dll1/Dll4 Notch ligands during acute GVHD. Combined Dll1/Dll4 blockade was as potent as DNMAML expression in decreasing cytokine production by alloreactive T cells, demonstrating that Delta-like and not Jagged ligands are the key Notch agonists at the alloimmune synapse. Dll4 inhibition was superior to Dll1 blockade in reducing cytokine production, abrogating GVHD, and prolonging recipient survival. Importantly, combined Dll1/Dll4 inhibition provided long-term protection against GVHD morbidity and mortality, while avoiding severe gastrointestinal side effects from Notch inhibition. Protection was observed even upon transient Dll1/Dll4 blockade during 1–2 weeks after transplantation. Altogether, our data suggest that Notch1 and Dll4 preferentially interact during alloreactive T cell priming and identify novel strategies to safely and efficiently target individual elements of the Notch pathway after allo-BMT. Humanized antibodies against Notch receptors and ligands were designed to block both mouse and human proteins, thus our preclinical work could lead to new strategies for GVHD control in human patients. Disclosures: Shelton: Genentech Inc.: Employment. Yan:Genentech Inc.: Employment. Siebel:Genentech Inc.: Employment.

scholarly journals Delta-like Ligands Expressed By Stromal Cells in Secondary Lymphoid Organs Deliver an Early Pulse of Notch Signaling and Drive T Cell Pathogenicity in Acute Graft-Versus-Host Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 841-841
Author(s):  
Jooho Chung ◽  
Christen L. Ebens ◽  
Vedran Radojcic ◽  
Ute Koch ◽  
Ann Friedman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Notch Signaling ◽  
Stromal Cells ◽  
Acute Gvhd ◽  
Hematopoietic System ◽  
Alloreactive T Cells ◽  
Equity Ownership

Abstract Notch signaling is a critical regulator of T cell effector functions during acute graft-versus-host disease (GVHD). Pan-Notch inhibition in donor-derived T cells or systemic antibody-mediated blockade of Delta-like1 (Dll1) and Delta-like4 (Dll4) Notch ligands results in near-complete protection from acute GVHD in mouse models of allogeneic bone marrow transplantation. Notch-deprived alloreactive T cells proliferate and accumulate in vivo, but produce dramatically reduced levels of the proinflammatory cytokines IFNγ, TNFα and interleukin-2 (IL-2) (Zhang et al., Blood 2011; Sandy et al., J Immunol 2013; Tran et al., J Clin Invest 2013). In this study, we sought to: 1) determine the kinetic requirements for Notch signaling in the pathogenesis of acute GVHD; 2) identify the essential cellular compartment that delivers Dll1 and/or Dll4 ligands to incoming alloreactive T cells. In the B6 anti-BALB/c major histocompatibility complex-mismatched model, a single dose of Dll1 and Dll4 blocking antibodies at the time of transplantation abolished alloreactive T cell production of IFNγ, TNFα, and IL-2, increased regulatory T cell numbers (as assessed at day 10), and conferred long-term protection from GVHD. Conversely, delaying antibody administration by only two days after transplantation resulted in persistent T cell cytokine production, no changes in regulatory T cell numbers, and loss of long-term protection from GVHD. These findings identify a critical early window of Notch activity that promotes the pathogenesis of acute GVHD. To identify the dominant cellular source of Dll1 and Dll4, we assessed the impact of Cre-mediated Dll1 and Dll4 inactivation within host hematopoietic, donor hematopoietic, or host non-hematopoietic tissues. Bone marrow chimeras that lacked Dll1 and Dll4 solely within the host hematopoietic system were generated from poly(I:C)-induced Mx1-Cre;Dll1fl/fl;Dll4fl/fl donor mice. Both donor chimerism and Cre-mediated excision efficiency were >97%. Unlike systemic Dll1/4 blockade, Dll1 and Dll4 inactivation within the host hematopoietic system failed to decrease GVHD mortality or severity. Likewise, Mx1-Cre-mediated deletion of Dll1 and Dll4 within the donor hematopoietic system had minimal effects on T cell proinflammatory cytokines. In contrast, Ccl19-Cre-mediated Dll1 and Dll4 inactivation within host stromal cells profoundly impaired donor T cell production of IFNγ, TNFα, and IL-2, and resulted in long-term protection from GVHD. Lineage tracing in Ccl19-Cre x ROSA26-YFP mice revealed Cre activity within a small subset of CD45-negative lymph node and spleen stromal cells, but not in professional hematopoietic antigen-presenting cells. These data suggest that a specialized subset of non-hematopoietic stromal cells delivers an early pulse of Notch signaling to alloreactive T cells during acute GVHD. To our knowledge, these results provide the first in vivo evidence for non-motile secondary lymphoid-resident stromal cells as critical drivers of T cell-mediated immune pathology, with a central role for Notch signaling in this process. Transient interference with Notch ligand function or with their expression by the stromal cell niche in the peri-transplant period could serve as a novel therapeutic strategy for GVHD. Disclosures Yan: Genentech: Employment, Equity Ownership. Siebel:Genentech: Employment, Equity Ownership.

Notch Inhibition in Alloreactive CD4+ and CD8+ T Cells Blocks Graft-Versus-Host Disease by Inducing a Hyporesponsive Program with Features of T Cell Anergy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 340-340
Author(s):  
Ashley R Sandy ◽  
Jooho Chung ◽  
Ivy T Tran ◽  
Gloria T Shan ◽  
Ann Friedman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Notch Signaling ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Ifn Gamma ◽  
Cell Compartments ◽  
Alloreactive T Cells ◽  
Notch Inhibition

Abstract Abstract 340 Graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality following allogeneic bone marrow transplantation (allo-BMT). We previously identified Notch signaling as an essential regulator of allogeneic CD4+ T cell responses mediating GVHD after allo-BMT. Alloreactive CD4+ T cells expressing the pan-Notch inhibitor DNMAML induced markedly less severe GVHD as compared to wild-type T cells, leading to improved survival of the recipients. Notch-deprived T cells had preserved in vivo expansion and cytotoxicity. However, alloreactive DNMAML CD4+ T cells produced markedly decreased amounts of multiple proinflammatory cytokines, including TNF-alpha, IFN-gamma, and IL-2. This was associated with increased expansion of Foxp3+ CD4+ T regulatory cells. Thus, Notch signaling is an attractive new therapeutic target to control GVHD without eliminating the anti-cancer activity of allo-BMT. To elucidate the mechanisms of Notch action in GVHD, we studied the effects of Notch inhibition in alloreactive CD4+ and CD8+ T cells using minor and major histocompatibility antigen-mismatched models of allo-BMT. In the B6 anti-BALB/b minor antigen-mismatched model, recipients of B6 T cells were protected from lethal acute GVHD upon DNMAML expression in the CD4+, CD8+ or both T cell compartments. In the B6 anti-BALB/c MHC-mismatched model, DNMAML CD4+ or CD8+ T cells transplanted alone or in combination induced significantly less GVHD and resulted in improved survival compared to wild-type T cells. Upon ex vivo restimulation with anti-CD3/CD28 antibodies, both CD4+ and CD8+ DNMAML alloreactive T cells had markedly decreased production of IFN-gamma. These findings suggest that Notch signaling has parallel functions in CD4+ and CD8+ T cells. We then studied expression of Tbx21 (encoding T-bet) and Eomes, the key transcription factors regulating Ifng transcription in CD4+ Th1 and CD8+ T cells, respectively. DNMAML alloreactive T cells had preserved amounts of Tbx21 mRNA and T-bet protein, and increased levels of Eomes transcripts and protein. These data differ from past reports indicating that Notch signaling controls T cell differentiation through direct regulation of Tbx21 and Eomes expression. Ex vivo restimulation of DNMAML CD4+ and CD8+ T cells with PMA (diacylglycerol analog) and ionomycin (calcium ionophore) rescued IFN-gamma production by both T cell compartments and partially restored IL-2 production by CD4+ T cells, suggesting abnormal signaling downstream of the T cell receptor. After anti-CD3/CD28 restimulation, DNMAML alloreactive T cells showed markedly decreased phosphorylation of Mek1 and Erk1/2, indicating defective Ras/MAPK activation. PMA was sufficient to rescue Erk1/2 activation. NFkB activity was also significantly impaired in alloreactive DNMAML T cells as assessed with a NFkB-luciferase reporter transgene. Abnormal responsiveness was acquired in vivo during alloreactive T cell priming, since naïve DNMAML T cells had preserved Ras/MAPK activation. Moreover, alloreactive Notch-deprived T cells had elevated levels of intracellular cAMP and increased expression of the anergy-associated genes, Dgka and Egr3. Thus, alloreactive DNMAML T cells had features reminiscent of T cell anergy. Given that in vivo proliferation in irradiated recipients and cytotoxicity of DNMAML alloreactive T cells were largely preserved, our data suggest a “split anergy” phenotype with differential effects on distinct T cell effector functions. Altogether, our results reveal a parallel role for Notch signaling in both the CD4+ and CD8+ T cell compartments that differ from all previous reports of Notch action in mature T cells. Understanding the role of Notch signaling in alloreactive T cells is essential for harnessing the therapeutic potential of Notch inhibition in GVHD. Disclosures: No relevant conflicts of interest to declare.

Anergic T cells exert antigen-independent inhibition of cell-cell interactions via chemokine metabolism

Blood ◽  
2003 ◽  
Vol 102 (6) ◽  
pp. 2173-2179 ◽  
Author(s):  
Martha J. James ◽  
Lavina Belaramani ◽  
Kanella Prodromidou ◽  
Arpita Datta ◽  
Sussan Nourshargh ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
T Cell Activation ◽  
Cell Interactions ◽  
Cell Surface Expression ◽  
Anergic T Cells ◽  
Parenchymal Cells ◽  
Cell Cell

Abstract Due to their ability to inhibit antigen-induced T-cell activation in vitro and in vivo, anergic T cells can be considered part of the spectrum of immunoregulatory T lymphocytes. Here we report that both murine and human anergic T cells can impair the ability of parenchymal cells (including endothelial and epithelial cells) to establish cell-cell interactions necessary to sustain leukocyte migration in vitro and tissue infiltration in vivo. The inhibition is reversible and cell-contact dependent but does not require cognate recognition of the parenchymal cells to occur. Instrumental to this effect is the increased cell surface expression and enzymatic activity of molecules such as CD26 (dipeptidyl-peptidase IV), which may act by metabolizing chemoattractants bound to the endothelial/epithelial cell surface. These results describe a previously unknown antigen-independent anti-inflammatory activity by locally generated anergic T cells and define a novel mechanism for the long-known immunoregulatory properties of these cells.

Reduction of TGF-beta activity abrogates growth promoting tumor cell-cell interactions in vivo

1991 ◽  
Vol 148 (3) ◽  
pp. 380-390 ◽  
Author(s):  
D. Theodorescu ◽  
M. Caltabiano ◽  
R. Greig ◽  
D. Rieman ◽  
R. S. Kerbel
Keyword(s):  
Tumor Cell ◽  
Cell Interactions ◽  
Beta Activity ◽  
Tgf Beta ◽  
Growth Promoting ◽  
Cell Cell

scholarly journals Formation of organotypic testicular organoids in microwell culture†

2019 ◽  
Vol 100 (6) ◽  
pp. 1648-1660 ◽  
Author(s):  
Sadman Sakib ◽  
Aya Uchida ◽  
Paula Valenzuela-Leon ◽  
Yang Yu ◽  
Hanna Valli-Pulaski ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Germ Cell ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Cell Interactions ◽  
Dependent Manner ◽  
Tissue Architecture ◽  
Cell Cell

Abstract Three-dimensional (3D) organoids can serve as an in vitro platform to study cell–cell interactions, tissue development, and toxicology. Development of organoids with tissue architecture similar to testis in vivo has remained a challenge. Here, we present a microwell aggregation approach to establish multicellular 3D testicular organoids from pig, mouse, macaque, and human. The organoids consist of germ cells, Sertoli cells, Leydig cells, and peritubular myoid cells forming a distinct seminiferous epithelium and interstitial compartment separated by a basement membrane. Sertoli cells in the organoids express tight junction proteins claudin 11 and occludin. Germ cells in organoids showed an attenuated response to retinoic acid compared to germ cells in 2D culture indicating that the tissue architecture of the organoid modulates response to retinoic acid similar to in vivo. Germ cells maintaining physiological cell–cell interactions in organoids also had lower levels of autophagy indicating lower levels of cellular stress. When organoids were treated with mono(2-ethylhexyl) phthalate (MEHP), levels of germ cell autophagy increased in a dose-dependent manner, indicating the utility of the organoids for toxicity screening. Ablation of primary cilia on testicular somatic cells inhibited the formation of organoids demonstrating an application to screen for factors affecting testicular morphogenesis. Organoids can be generated from cryopreserved testis cells and preserved by vitrification. Taken together, the testicular organoid system recapitulates the 3D organization of the mammalian testis and provides an in vitro platform for studying germ cell function, testicular development, and drug toxicity in a cellular context representative of the testis in vivo.

scholarly journals The Molecular Landscape and Essential Pathways Involved in Bone Marrow-Mediated Carfilzomib Resistance of Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1571-1571
Author(s):  
Jonas Schwestermann ◽  
Andrej Besse ◽  
Lenka Besse ◽  
Christoph Driessen
Keyword(s):  
Stromal Cells ◽  
Cytokine Signaling ◽  
Cell Contact ◽  
Library Screening ◽  
Nsg Mice ◽  
Molecular Landscape ◽  
Rpmi 8226 ◽  
Cell Cell

Abstract Background Multiple myeloma (MM) remains an incurable malignancy, with most patients relapsing and dying from the disease. Anti-myeloma drugs, such as proteasome inhibitors (PIs) bortezomib and carfilzomib (CFZ), have considerably improved prognosis in myeloma. Despite these advances, disease heterogeneity, early relapse and treatment resistance still pose major challenges in MM treatment. Understanding the mechanisms that mediate PI resistance provide a key to targeting both, PI-resistant minimal residual disease that drives relapsed MM after prolonged PI-containing frontline therapy, as well as PI-refractory, aggressive advanced MM. While key mechanisms of the in vitro-generated PI resistance in MM have been revealed in cell line models, we lack understanding of PI resistance in vivo, where in particular clonal heterogeneity and the tumor microenvironment (TME) within the bone marrow (BM) add additional levels of complexity. Therefore, the aim of our study was to analyze the molecular landscape and changes occurring during MM progression under CFZ treatment in vivo and to identify key molecular processes contributing to CFZ-resistance of MM cells in the presence of stromal cells in vitro, to ultimately identify new molecular pathways and develop innovative treatment strategies in PI-resistant MM. Methods The NSG mice intrafemorally engrafted with human RPMI-8226 cells were either untreated or treated long-term with 4 mg/kg CFZ (intravenously) until they became drug resistant. At this point, CFZ naïve and CFZ-resistant cells were isolated and processed for single-cell RNA sequencing (scRNA-seq, 10x Genomics) with the aim to characterize a transcriptional CFZ-resistance signature in refractory cells. To investigate the role of the TME as well as the importance of cell-cell interactions in CFZ-resistance in vitro, we performed two independent genome-wide CRISPR/Cas9 library screenings. In the first one, Brunello library transduced RPMI-8226 cells were co-cultured with human stromal cells (HS5) and treated with CFZ to identify CFZ sensitivity/resistance candidate genes. In the second experiment, Brunello library and synthetic Notch (synNotch) receptor transduced HS5 cells were co-cultured with synNotch ligand transduced RPMI-8226 cells to identify genes that are essential for establishing cell-cell contacts between stromal and MM cells. Subsequent functional analysis of the highest-ranking CFZ sensitivity/resistance candidates in the RPMI-8226+HS5 co-culture included shRNA-silencing, single-gene knockouts, viability assays, cell cycle analysis and protein synthesis analysis using the SUnSET assay. Results ScRNA-seq analysis of CFZ-refractory RPMI-8226 cells growing in the BM of NSG mice showed a different transcriptional landscape, compared to CFZ-naïve cells isolated from the BM of untreated mice. The unsupervised clustering analysis, using UMAP, revealed that cells exposed to CFZ show distinct populations with a strong increase in the OXPHOS and protein folding capacity as well as down-regulation of several genes involved in proliferation and apoptosis, when compared to naïve cells. The CRISPR/Cas9 library screening where RPMI-8226 cells were co-cultured with HS5 cells and exposed to CFZ revealed several CFZ sensitivity candidates at the cut-off of false discovery rate (FDR) &lt; 0.01 and fold change above 1.5-fold. Those genes are involved in cytokine signaling, cell growth, invasion, metastasis and quality control of translational elongation. At the same time, the CRISPR/Cas9 library screening, where synNotch receptor transduced HS5 cells were co-cultured with synNotch ligand transduced RPMI-8226 cells revealed gene candidates at the cut-off of FDR &lt; 0.01 and fold change greater than 1.5-fold, which mediate stronger or weaker cell-cell interaction. Those genes are particularly involved in cytokine signaling and mitochondrial metabolism. Conclusion In conclusion, MM cells that acquired CFZ-resistance upon cell-cell contact with certain cell types within the TME, such as stromal cells, differ significantly from CFZ-naïve cells. CFZ-resistance, caused by cell-cell contact with stromal cells, is presumably mediated via decreased proliferative as well as protein synthesis capacity of MM cells. Therefore, stimulation of MM cells to proliferate and synthesize more proteins may be a key to targeting CFZ-resistance in vivo. Disclosures No relevant conflicts of interest to declare.

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