220 THE ESTROGEN-LIKE EFFECT OF 2-METHOXYESTRADIOL (2-ME) IN IN VITRO AND IN VIVO MODELS

2015 ◽  
Vol 27 (1) ◽  
pp. 200
Author(s):  
J.-S. Lee ◽  
E.-B. Jeung
Keyword(s):  
Mrna Expression ◽  
Therapeutic Effects ◽  
Mrna Levels ◽  
Oestrogen Receptors ◽  
In Vivo Models ◽  
Ru 486 ◽  
Final Injection ◽  
M 10

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17β-oestradiol, interacts with oestrogen receptors and microtubules and has a low affinity for oestrogen receptors (ER). It has attracted considerable interest due to its potential anti-cancer therapeutic effects. 2-ME is also recognised for its unique and profound actions on various tumour cell lines and cancer independent of the hormone receptor status. Regardless of differences in function, 2-ME has an affinity for ER, however, the exact mechanisms of 2-ME action via the ER are not fully understood. In the current study, we examined the estrogenic effect of 2-ME on mRNA levels of CaBP-9k, ER, and progesterone receptor (PR) in the absence or presence of the 17β-oestradiol (E2) and progesterone (P4) in both in vivo and in vitro models by real-time RT–PCR. In vitro, cells (n = 3 per group) were exposed to a single dose of E2 (10–9 M), P4 (10–6 M), 2-ME (10–8 M, 10–7 M, 10–6 M). The mechanism of CaBP-9k induction by these chemicals pre-treated with 10–7 M ICI 182, 780 and 10–6 M RU 486 for 30 min before exposure to E2 and 2-ME were analysed. In vivo, 35 female ICR mice (PND 14 days) were divided into 7 groups (n = 5 per group), and each group was administered subcutaneously with 24% DMSO, 38% ethanol, and 38% sterile saline as a vehicle, E2 [40 μg kg–1 of body weight (BW)] a physiological dose level), 2-ME (4, 40, and 80 mg kg–1 of BW) for 3 days. The mice were killed 24 h after the final injection. To investigate the effect of antagonism, 10 mice were injected SC with ICI 182 780 (10 mg kg–1 of BW) and RU 486 (10 mg kg–1 of BW) at 30 min before injection with 2-ME (40 mg kg–1 of BW) for 3 days and killed 24 h after the final injection. Results are presented as mean ± s.e.m.; P-values were calculated using one-way ANOVA. In GH3 cells, the mRNA level of CaBP-9k was induced in the E2 (10–9 M) treatment group, and expression of CaBP-9k was also up-regulated in the 2-ME (10–7 M)-treated group. Uterine lactoferrin (Ltf) mRNA expression was also increased in the 2-ME (40 mg kg–1 of BW) group, similar to the response with E2 (40 μg kg–1 of BW) in mice. As a blocker for ER and PR activity, ICI 182 780 and RU 486 reversed the E2 or 2-ME mediated increase of CaBP-9k and Ltf mRNA expression. We found that 2-ME significantly increased the levels of ERa and PR transcripts. In parallel with in vitro results, the mRNA levels of ERa and PR were induced by treatment with E2 and 2-ME. Taken together, our findings demonstrated that expression of estrogenic markers, CaBP-9k and Ltf, was regulated by 2-ME in both in vitro and in vivo, which may increase their estrogenic activities in female during the cycle through ER and/or PR-mediated pathway.

scholarly journals Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hai-Jun Gao ◽  
Xu-Dong Sun ◽  
Yan-Ping Luo ◽  
Hua-Sheng Pang ◽  
Xing-Ming Ma ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mrna Expression ◽  
Echinococcus Granulosus ◽  
Calcium Content ◽  
Mrna Levels ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein

Abstract Background Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. Methods The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. Results In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 μg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. Conclusions Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

Annexin V decreases PS-mediated macrophage efferocytosis and deteriorates elastase-induced pulmonary emphysema in mice

2012 ◽  
Vol 303 (10) ◽  
pp. L852-L860 ◽  
Author(s):  
S. Yoshida ◽  
N. Minematsu ◽  
S. Chubachi ◽  
H. Nakamura ◽  
M. Miyazaki ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Apoptosis ◽  
Pulmonary Emphysema ◽  
Keratinocyte Growth Factor ◽  
Annexin V ◽  
Phenotypic Change ◽  
Chronic Obstructive ◽  
Mrna Levels

Efferocytosis is believed to be a key regulator for lung inflammation in chronic obstructive pulmonary disease. In this study we pharmacologically inhibited efferocytosis with annexin V and attempted to determine its impact on the progression of pulmonary emphysema in mouse. We first demonstrated in vitro and in vivo efferocytosis experiments using annexin V, an inhibitor for phosphatidylserine-mediated efferocytosis. We then inhibited efferocytosis in porcine pancreatic elastase (PPE)-treated mice. PPE-treated mice were instilled annexin V intranasally starting from day 8 until day 20. Mean linear intercept (Lm) was measured, and cell apoptosis was assessed in lung specimen obtained on day 21. Cell profile, apoptosis, and mRNA expression of matrix metalloproteinases (MMPs) and growth factors were evaluated in bronchoalveolar lavage (BAL) cells on day 15. Annexin V attenuated macrophage efferocytosis both in vitro and in vivo. PPE-treated mice had a significant higher Lm, and annexin V further increased that by 32%. More number of macrophages was found in BAL fluid in this group. Interestingly, cell apoptosis was not increased by annexin V treatment both in lung specimens and BAL fluid, but macrophages from mice treated with both PPE and annexin V expressed higher MMP-2 mRNA levels and had a trend for higher MMP-12 mRNA expression. mRNA expression of keratinocyte growth factor tended to be downregulated. We showed that inhibited efferocytosis with annexin V worsened elastase-induced pulmonary emphysema in mice, which was, at least partly, attributed to a lack of phenotypic change in macrophages toward anti-inflammatory one.

scholarly journals Effect of the proinflammatory cytokine TNF-α on intestinal riboflavin uptake: inhibition mediated via transcriptional mechanism(s)

2018 ◽  
Vol 315 (5) ◽  
pp. C653-C663 ◽  
Author(s):  
Kasin Yadunandam Anandam ◽  
Omar A. Alwan ◽  
Veedamali S. Subramanian ◽  
Padmanabhan Srinivasan ◽  
Rubina Kapadia ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Significant Inhibition ◽  
Mrna Levels ◽  
In Vivo Models ◽  
Uptake Inhibition ◽  
Tnf Α ◽  
Vitro Model ◽  
Factor Α

Riboflavin (RF), is essential for normal cellular metabolism/function. Intestinal RF absorption occurs via a specific carrier-mediated process that involves the apical transporter RFVT-3 ( SLC52A3) and the basolateral RFVT-1 (SLC52A1). Previously, we characterized different cellular/molecular aspects of the intestinal RF uptake process, but nothing is known about the effect of proinflammatory cytokines on the uptake event. We addressed this issue using in vitro, ex vivo, and in vivo models. First, we determined the level of mRNA expression of the human (h)RFVT-3 and hRFVT-1 in intestinal tissue of patients with inflammatory bowel disease (IBD) and observed a markedly lower level compared with controls. In the in vitro model, exposing Caco-2 cells to tumor necrosis factor-α (TNF-α) led to a significant inhibition in RF uptake, an effect that was abrogated upon knocking down TNF receptor 1 (TNFR1). The inhibition in RF uptake was associated with a significant reduction in the expression of hRFVT-3 and -1 protein and mRNA levels, as well as in the activity of the SLC52A3 and SLC52A1 promoters. The latter effects appear to involve Sp1 and NF-κB sites in these promoters. Similarly, exposure of mouse small intestinal enteroids and wild-type mice to TNF-α led to a significant inhibition in physiological and molecular parameters of intestinal RF uptake. Collectively, these findings demonstrate that exposure of intestinal epithelial cells to TNF-α leads to inhibition in RF uptake and that this effect is mediated, at least in part, via transcriptional mechanism(s). These findings may explain the significantly low RF levels observed in patients with IBD.

scholarly journals In Vitro and In Vivo Models of Cerebral Ischemia Show Discrepancy in Therapeutic Effects of M2 Macrophages

PLoS ONE ◽  
2013 ◽  
Vol 8 (6) ◽  
pp. e67063 ◽  
Author(s):  
Virginie Desestret ◽  
Adrien Riou ◽  
Fabien Chauveau ◽  
Tae-Hee Cho ◽  
Emilie Devillard ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Therapeutic Effects ◽  
M2 Macrophages ◽  
In Vivo Models

scholarly journals The Ashwell-Morell Receptor Regulates Hepatic Thrombopoietin Production Via JAK2-STAT3 Signaling in Vivo and in Vitro

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2-2
Author(s):  
Renata Grozovsky ◽  
Antonija Jurak Begonja ◽  
John H. Hartwig ◽  
Herve Falet ◽  
Karin M Hoffmeister
Keyword(s):  
Bone Marrow ◽  
Sialic Acid ◽  
Mrna Expression ◽  
Hepg2 Cells ◽  
Mrna Levels ◽  
Platelet Production ◽  
Platelet Survival ◽  
Post Infusion

Abstract The human body produces and removes 1011 platelets daily to maintain a normal steady-state platelet count, and the level of production can be greatly increased under conditions of platelet destruction. Thrombopoietin (TPO) is the primary regulator of platelet production, supporting the survival, proliferation and differentiation of platelet precursors, bone marrow megakaryocytes. Hepatocytes are a major source of production and secretion of circulating TPO. However, mechanisms regulating circulating TPO levels have been debated for decades. Here, we provide experimental evidence that platelets lacking sialic acid (desialylated platelets) are removed by the hepatic Ashwell-Morell receptor (AMR or asialoglycoprotein receptor), thereby regulating platelet survival and hepatic TPO levels. These conclusions are based on the following evidence: 1) Mice lacking the AMR Asgr2 subunit had increased platelet survival, compared to wild type (WT) mice. Platelets from Asgr2-null mice showed increased loss of sialic acid, as evidenced by flow cytometry using the galactose specific lectins RCAI and ECL, showing that removal of desialylated platelets by the AMR regulates in vivo platelet survival. 2) Livers isolated from Asgr2-null mice had TPO mRNA levels decreased by 40%, compared to WT mice. In contrast, liver TPO mRNA levels were increased by 30% in St3gal4-null mice lacking the sialyltransferase ST3GalIV, where desialylated platelet clearance is increased and specifically mediated by the AMR. Both plasma TPO levels and platelet TPO contents were similarly altered in both mutant mice. Thus, desialylated platelet uptake by the AMR regulated liver TPO levels. 3) Desialylated platelets isolated from St3gal4-null or Asgr2-null mice infused into WT mice increased hepatic TPO mRNA levels as early as 12h post-infusion. Plasma TPO concentrations and bone marrow megakaryocyte numbers increased in parallel with TPO mRNA levels, peaking by day 2 post-infusion, followed by new platelet release at day 10 post-infusion. In contrast, desialylated platelets infused into Asgr2-null mice had no effect on TPO mRNA synthesis, TPO plasma levels and bone marrow megakaryocyte numbers. 4) Incubation of human hepatoma cell line, HepG2 cells, with human desialylated platelets by sialidase treatment resulted in TPO mRNA expression increase by 2.2 and 2.9-fold after 4 and 6h, respectively, followed by significant increase in TPO secretion. 5) The signaling pathways activated by uptake of desialylated platelets by the AMR to induce TPO mRNA transcription were investigated in vivo and in vitro. Major polypeptides of 60-70 and 125 kDa were highly tyrosine phosphorylated in WT liver cells, as evidenced by SDS-PAGE. Using a specific antibody directed against JAK2, we identified the 125-kDa phosphoprotein as the tyrosine kinase JAK2 in mouse liver cells and human HepG2 cells. Analysis of liver samples revealed a marked reduction in JAK2 phosphorylation in Asgr2-null mice and significant increase in St3gal4-null mice. 6) The JAK1/2 inhibitor AZD1480 significantly decreased phosphorylation of JAK2, phosphorylation and translocation to the nucleus of the acute phase response transcription factor STAT3, TPO mRNA expression and TPO secretion in HepG2 cells incubated with desialylated platelets. In vivo treatment of WT mice with AZD1480 blocked TPO mRNA increase promoted by injection of endogenously desialylated platelets. Therefore we conclude that platelets desialylate as they circulate, thereby becoming the primary AMR ligand and providing a novel physiological feedback mechanism to regulate plasma TPO levels and platelet production in vivo and in vitro. Disclosures No relevant conflicts of interest to declare.

scholarly journals Anti-Psoriasis Effects and Mechanisms of Α-(8-Quinolinoxy) Zinc Phthalocyanine-Mediated Photodynamic Therapy

2017 ◽  
Vol 44 (1) ◽  
pp. 200-214 ◽  
Author(s):  
Han-Qing Liu ◽  
Ying-Ming Wang ◽  
Wan-Fang Li ◽  
Chao Li ◽  
Zhi-Huan Jiang ◽  
...  
Keyword(s):  
Photodynamic Therapy ◽  
Mrna Expression ◽  
T Lymphocyte ◽  
B Cell Lymphoma ◽  
Zinc Phthalocyanine ◽  
Polymerase Chain Reaction Test ◽  
Therapeutic Effects ◽  
Hacat Cells

Background/Aims: The aim of this study was to determine the anti-psoriasis effects of α-(8-quinolinoxy) zinc phthalocyanine (ZnPc-F7)-mediated photodynamic therapy (PDT) and to reveal its mechanisms. Methods: HaCaT cells were used to observe the influence of ZnPc-F7-PDT on cell proliferation in vitro. The in vivo anti-psoriasis effects of ZnPc-F7-PDT were evaluated using a mouse vagina model, a propranolol-induced cavy psoriasis model and an imiquimod (IMQ)-induced nude mouse psoriasis model. Flow cytometry was carried out to determine T lymphocyte levels. Western blotting was performed to determine protein expression, and a reverse transcription-polymerase chain reaction test was performed to determine mRNA expression. Results: The results showed that ZnPc-F7-PDT significantly inhibited the proliferation of HaCaT cells in vitro; when the light doses were fixed, changing the irradiation time or output power had little influence on the inhibition rate. ZnPc-F7-PDT significantly inhibited the hyperproliferation of mouse vaginal epithelium induced by diethylstilbestrol and improved propranolol- and IMQ-induced psoriasis-like symptoms. ZnPc-F7-PDT inhibited IMQ-induced splenomegaly and T lymphocyte abnormalities. ZnPc-F7-PDT did not appear to change T lymphocytes in the mouse vagina model. ZnPc-F7-PDT down-regulated the expression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (Bcl-2), interleukin (IL)-17A mRNA and IL-17F mRNA, and up-regulated the expression of Bax. Conclusion: In conclusion, ZnPc-F7-PDT exhibited therapeutic effects in psoriasis both in vitro and in vivo and is a potential approach in the treatment of psoriasis. Potential mechanisms of these effects included the inhibition of hyperproliferation; regulation of PCNA, Bcl-2, Bax, IL-17A mRNA and IL-17F mRNA expression; and immune regulation.

scholarly journals Steroid-sensitive gene-1 is an androgen-regulated gene expressed in prostatic smooth muscle cells in vivo

2001 ◽  
Vol 26 (3) ◽  
pp. 175-184 ◽  
Author(s):  
D Marcantonio ◽  
LE Chalifour ◽  
MA Alaoui-Jamali And H T Huynh ◽  
MA Alaoui-Jamali ◽  
MA Alaoui-Jamali ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Mrna Expression ◽  
Muscle Cells ◽  
Ventral Prostate ◽  
Mrna Levels ◽  
Rat Ventral Prostate ◽  
Steroid Sensitive

Steroid-sensitive gene-1 (SSG1) is a novel gene we cloned, found regulated by 17beta-estradiol in the rat uterus and mammary gland, and over-expressed in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. We show here that SSG1 mRNA and protein expression are regulated by androgens in the rat ventral prostate. Increases in SSG1 mRNA levels were detected by Northern blotting after 24 h and reached a 27-fold peak 96 h following castration, relative to SSG1 mRNA expression in sham-operated rats. Dihydrotestosterone or testosterone supplementation of castrated rats prevented this rise in SSG1 mRNA. In contrast with SSG1 mRNA expression, SSG1 protein was decreased 16-fold 2 weeks following castration but was at control levels in the prostates of castrated rats receiving dihydrotestosterone or testosterone. Although SSG1 is regulated by androgens in vivo, treatment of LnCap cells with dihydrotestosterone, cyproterone acetate or flutamide did not result in the regulation of SSG1 protein levels in vitro. Immunofluorescence studies show that SSG1 is mainly expressed in prostatic smooth muscle cells. These results indicate that SSG1 is an androgen-regulated gene that is expressed in the smooth muscle component of the rat ventral prostate in vivo.

Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles

2013 ◽  
Vol 25 (8) ◽  
pp. 1194 ◽  
Author(s):  
G. L. Vasconcelos ◽  
M. V. A. Saraiva ◽  
J. J. N. Costa ◽  
M. J. Passos ◽  
A. W. B. Silva ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Nuclear Antigen ◽  
Mrna Levels ◽  
Related Factors ◽  
Differentiation Factor ◽  
Growth Differentiation Factor 9 ◽  
Before And After ◽  
Vitro Development

The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e. hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL–1 from Days 0 to 6 and 500 ng mL–1 from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P < 0.05). Alone, 200 ng mL–1 GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL–1 bovine serum albumin, 10 µg mL–1 insulin, 5.5 µg mL–1 transferrin, 5 ng mL–1 selenium, 2 mM glutamine, 2 mM hypoxanthine and 50 μg mL–1 ascorbic acid (α-MEM+). Comparisons of uncultured (0.2 mm) and α-MEM+ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P < 0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.

scholarly journals Mechanisms Regulating Myeloma Cell Responsiveness to Antisense Oligonucleotides Targeting IRF4

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-32
Author(s):  
Ryosuke Shirasaki ◽  
Esperanza M Algarín ◽  
Ricardo De Matos Simoes ◽  
Sondra L. Downey-Kopyscinski ◽  
Shizuka Yamano ◽  
...  
Keyword(s):  
High Risk ◽  
Cell Lines ◽  
Cell Response ◽  
Antisense Oligonucleotides ◽  
Research Funding ◽  
Mrna Levels ◽  
In Vivo Models ◽  
Genome Scale

Functional genomics studies from our group and others, including CRISPR screens, have documented that interferon regulatory factor 4 (IRF4) is a critical transcription factor (TF) for multiple myeloma (MM) cells in preclinical in vitro and in vivo models; and one of the top most pronounced and recurrent dependencies for MM cells vs. other neoplasias. IRF4 lacks known ligand-binding pocket(s) amenable to selective inhibition by small-molecule pharmacological agents. IRF4 is thus considered, as many TFs, as "undruggable". Recent progress in antisense oligonucleotides (ASOs) and their in vivo properties motivated us to systematically evaluate the response of MM cells to anti-IRF4 ASOs, to obtain direct insights into their potential therapeutic applications in MM and also as functional probes into IRF4 biology. We observed that a panel of anti-IRF4 ASOs (but not control ASO) exhibited dose- and time-dependent activity against genotypically diverse MM cell lines (sub-uM IC50s for most MM lines vs. &gt;10uM against non-MM cells lacking IRF4, e.g. HS5 bone marrow stromal cells [BMSCs]). Major in vitro anti-MM activity could be observed within 3-5 days of treatment; and was preceded by significant decrease in intracellular IRF4 protein levels. Importantly, 24-hour exposure to IRF4 ASO (followed by washout and subsequent culture in ASO-free media for up to 6 days) can induce similar anti-MM activity as continuous in vitro exposure over 6 days. Combinations of IRF4-ASO with several established (e.g. proteasome inhibitor, thalidomide derivative, glucocorticoids) or investigational (e.g. venetoclax) agents for MM result led to enhanced or even synergistic effects in a panel of 6 MM cell lines. For some of these MM cell lines, their in vitro response to IRF4 ASO had statistically significant attenuation in co-cultures with BMSCs: this effect was partially recapitulated in MM cell monocultures supplemented by conditioned media from BMSCs or (to a lesser extent) rhIL6 treatment. To obtain insights into cell-autonomous and nonautonomous mechanisms regulating MM cell responses to IRF4 ASOs, we examined the transcriptional profiles of MM.1S cells exposed to ASOs; and also performed genome-scale CRISPR studies to define genes whose editing (loss-of-function, LOF) or activation (gain-of-function, GOF) alter the MM cell response to the IRF4 ASOs. The transcriptional signature of MM cell treatment with IRF4 ASO (vs. control ASO) comprised distinct clusters of downregulated genes with preferential expression in normal or malignant plasma cells vs. other lineages; CRISPR-validated roles as dependencies for MM cells in vitro (preferentially essential for MM vs. non-MM tumors; or pan-essential); and/or proximity to large areas of chromatin accessibility (defined by H3K27Ac genome-wide ChIP-Seq or ATAC-Seq). Importantly, several genes downregulated by IRF4 ASO treatment are upregulated in MM cells cocultured with BMSCs, possibly explaining at least in part the impact of co-cultures on anti-MM activity of ASO. In our integrated genome-scale CRISPR studies (and validation of many genes with individual sgRNAs), MM cell response to IRF4 ASOs was attenuated by LOF of endonuclease RNase H1 (RNASEH1; which degrades the target RNA when it establishes intracellular hybrids with the ASOs); LOF of diverse genes involved in ASO endocytosis (e.g. IGF2R, PICALM, SH3GL1, RAB5C) or regulation of chondroitin/heparan sulfate (SLC35B2, B3GAT3, B4GALT7, ALG5 and ALG6); GOF of IRF4 itself (likely reflecting a "stoichiometric" effect, e.g. induction of higher IRF4 mRNA levels may require higher concentrations of ASO to maintain anti-MM effect); and GOF of exocytosis-related genes(e.g. CLU, QPCT). Importantly, LOF or GOF of individual genes typically associated with high-risk MM was not associated with decreased MM cell response to the IRF4 ASOs: therefore, retaining intracellular accumulation of IRF4 ASO and productive knockdown of IRF4 mRNA remains a primary driver of anti-MM activity of ASOs even in the context of biologically aggressive MM cells with "high-risk" features. Our results provide comprehensive integrated assessment of the molecular and functional landscapes associated with dysregulation of IRF4; have direct implications for our mechanistic understanding of the role of IRF4 in MM biology; and provide a framework for IRF4 targeting by ASOs or potentially other therapeutic approaches. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Luo:Ionis Pharmaceuticals, Inc.: Current Employment. Kim:Ionis Pharmaceuticals, Inc.: Current Employment. MacLeod:Ionis Pharmaceuticals, Inc.: Current Employment. Mitsiades:Arch Oncology: Research Funding; Sanofi: Research Funding; FIMECS: Consultancy, Honoraria; Karyopharm: Research Funding; Abbvie: Research Funding; Takeda: Other: employment of a relative; TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; EMD Serono: Research Funding; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria.

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