Proteasome and HDAC Inhibitor Therapy in T-Cell Lymphoma (TCL) and Hodgkin Lymphoma (HL): Nuclear Factor Erythroid 2 like 2 (NRF2)-Dependent Cell Death and Function

Abstract Background: Activation of NF-kB pathways are a hallmark feature of TCL and HL, making proteasome inhibition an attractive therapeutic target. Previous studies have demonstrated prominent in vivo efficacy of ixazomib, an oral proteasome inhibitor for both TCL and HL. Among the common significantly regulated genes identified via systems biology approach include upregulation of genes encoding for ubiquitin proteasome subunits (Ravi et al. Cancer Res. 2016). Further combination studies with HDACi belinostat were synergistic in Jurkat, HH and L428 cell lines, and NRF2 was discovered as mediator of proteasome gene expression. We hypothesized that HDACi abrogates NRF2 mediated proteasome recovery leading to synergistic effects on cell viability in combination with ixazomib. Methods: Global transcriptome analysis was performed on RNA isolated from multiple cell lines include TCL (Jurkat) and HL (L540, L428) treated with ixazomib and control, as well as Jurkat cell lines treated with single agent ixazomib, belinostat and combination. Significant genes were determined by applying a one-way ANOVA with an adjusted Bonferroni correction for a false discovery rate (FDR) < 0.05. Further pathway analysis from significant genes was performed by using a fold change greater than ±1.2 comparing all samples to each other and observing pathway relationships using Ingenuity Pathway Analysis. Gene Set Enrichment Analysis was performed with FDR <0.05 for functional analysis. Proteasome-Glo cell based assay was used to evaluate caspase-, chymotrypsin, and trypsin-like activity. Proteasome activity was measured at 24 and 72 hours after Jurkat cell lines were treated with control, ixazomib or belinostat as single agents, and in combination. SiRNA knockdown experiments were performed in Jurkat cell line with NRF2 and non-targeting (NT) SiRNA transfection. Real-time quantitative PCR (qPCR) for proteasome subunit and NRF2 genes was performed on RNA isolated from treated cells. Results: Transcriptome analysis revealed upregulation of proteasome genes in ixazomib treated cell lines Jurkat, L540 and L428 at 24 hours. In Jurkat TCL, ixazomib caused decreased caspase-like and chymotrypsin-like proteasome activity at 24 hours that was followed by recovery of these activities at 72 hours. The combination of ixazomib and belinostat significantly decreased proteasome activity for chymotrypsin-like, caspase-like and trypsin-like activity at 72 hours compared to single agent ixazomib or belinostat. In Jurkat cells, NRF2 was identified as a transcriptional regulator involved in proteasome gene regulation, showing upregulation of proteasomal genes and NRF2 with ixazomib single agent, downregulation with belinostat single agent and in combination with ixazomib. These results were confirmed with qPCR for NRF2 and proteasome genes in Jurkat and L428. SiRNA knockdown for NRF2 in Jurkat cells resulted in decreased cell viability, NRF2 and proteasome gene expression compared with NT SiRNA following ixazomib treatment. Conclusions: Treatment with single agent ixazomib induced prominent proteasome gene expression in all TCL and HL cell lines. In Jurkat, recovery of chymotrypsin and caspase-like proteasome activity occurred by 72 hours suggesting that transcriptional changes induced by proteasome inhibition contributed to proteasome function recovery. Combination therapy with belinostat resulted in downregulation of proteasome genes in Jurkat and L428 and prevented functional recovery of the proteasome observed in Jurkat. Our results suggest that targeting the proteasome itself with ixazomib and preventing the induced recovery of proteasome genes with belinostat contributes to synergistic effects observed on proteasome function and cell viability in TCL and HL. Further studies with CRISPR/Cas to confirm the effect of NRF2 on proteasome gene and functional recovery in the context of proteasome inhibition are ongoing and will be reported. Disclosures Evens: Takeda: Other: Advisory board.

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Study on Leukemia Cell Differentiation and Drug Resistance By SATB1 Gene

Blood ◽

10.1182/blood.v124.21.5172.5172 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5172-5172

Author(s):

Chunyan Wang ◽

Huo Tan ◽

Liu Lei ◽

Lihua Xu

Keyword(s):

Gene Expression ◽

Drug Resistance ◽

Cell Differentiation ◽

Western Blot ◽

Cell Lines ◽

Jurkat Cell ◽

Leukemia Cell ◽

Jurkat Cells ◽

Leukemia Cells ◽

Chemotherapy Drugs

Abstract Background and objective: In this article, four types of lymphoma cell lines U937, Raji, Hut-102, Akata, and three types of leukemia cell lines K562, HL-60, Jurkat were used the expression of SATB1 in leukemia cells, Through cell differentiation induced by all-TRANS Retinoic acid and DMSO, finds the SATB1 gene expression is reduced, suggest that the SATB1 gene may be involved in cell differentiation. By gene silencing, reduce the SATB1 gene expression, sensitivity to chemotherapy drugs is increased, suggest that the SATB1 gene may be associated with drug resistance. Methods: 1. Western Blot Assay for detection of SATB1 gene expression in cell lines. 2. DMSO and ATRA to HL-60, Jurkat cells for different times, SATB1 gene expression in the cells are detected by Western Blot. 3. Building SATB1-shRNA, transfected Jurkat cells and to HL-60. 4. Testing inhibition rate of chemotherapy drugs on HL-60, HL-60-SATB1-ctr, HL-60-SATB1-sh, Jurkat, Jurkat-SATB1-ctr, Jurkat-SATB1-sh cell, suggesting its relationship with the drug resistance. Results: 1. SATB1 gene was expression in all the plant cells. 2. The expression of SATB1 in U937, Raji, Hut-102, Akata four types of lymphoma cell line is low. 3. It was highly expression in HL-60, Jurkat cells, but low in K562. 4. DMSO and ATRA treat HL-60, Jurkat cells for 48 and 96 hours, HL-60 cells become larger, rounded, Jurkat cells into smaller, is more obvious for 96-hour. 5. For HL-60, Jurkat cells, after DMSO and ATRA treated, the expression of SATB1 was decreased, more obvious for 96-hour. 6. HL-60-SATB1-sh1, Jurkat-SATB1-sh1 cell compared with the control group, the SATB1 protein content decreased significantly, successfully building SATB1-shRNA HL-60 and Jurkat cell line. 7. HL-60-SATB1-sh1, and Jurkat-SATB1-sh1 of daunorubicin (DNR) sensitivity has improved significantly. Conclusion: 1. The SATB1 gene expression in leukemia and lymphoma cells, and higher expression in leukemia cells. 2. After DMSO and ATRA treatment, the SATB1 expression significantly reduced, suggest that SATB1 may be involved in cell differentiation. 3. Successfully built SATB1-shRNA, and successfully transferred to HL-60 and Jurkat cell lines. 4. HL-60-SATB1-sh1, and Jurkat-SATB1-sh1 of daunorubicin (DNR) sensitivity increased significantly, prompting SATB1 may be related to drug-resistance. Disclosures No relevant conflicts of interest to declare.

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Changes in Mcl-1 and Bim Expression with Bortezomib and Melphalan Therapy for Multiple Myeloma.

Blood ◽

10.1182/blood.v106.11.2481.2481 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2481-2481 ◽

Cited By ~ 2

Author(s):

Rakesh Popat ◽

Lyndsey Goff ◽

Heather E. Oaekervee ◽

Jamie D. Cavenagh ◽

Simon P. Joel

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Viability ◽

Cell Lines ◽

Caspase 3 ◽

Proteasome Inhibition ◽

Myeloma Cell ◽

Jurkat Cells ◽

Cell Cycle Analysis ◽

Rpmi 8226

Abstract Background: Proteasome inhibition has been shown to be effective against a variety of tumours. In multiple myeloma the response rates to bortezomib (B) in relapsed patients is 46% (APEX study ≥ MR), but are likely to be up to 75% when combined with melphalan (M). Mechanisms underlying this effect are yet to be fully determined. Aims: To investigate the effect of B, M and the combination of the two on myeloma cell lines and primary patient cells, focusing on the anti-apoptotic molecule Mcl-1 and the pro-apoptotic molecule Bim in the mediation of drug activity. Cell cycle analysis using propidium iodide and flow cytometry was also performed in parallel. Methods: The human multiple myeloma cell lines (HMCLs) RPMI 8226/S, U266 and purified primary patient malignant plasma cells were used for cell culture and viability assays using an ATP bioluminescence method. Cells were incubated for 24 or 48 hours with differing concentrations of B, M and combinations in varying schedules. EC50 values were calculated using a sigmoidal Emax model and the observed cell viability of the combination of the two drugs was compared with the additive effect expected. Mcl-1, Bim, caspase-3, and PARP were probed for by Western Blotting of HMCLs. Results: Both U266 and RPMI 8226/S cells showed dramatic reductions in cell viability to B, with EC50 values of 4.7nM and 5.3nM respectively, and responded to high concentrations of M with EC50 values of 95.6 uM and 91.5 uM respectively after 48 hour incubations. Synergistic responses were seen when M was added 24 hours prior to B, but not with B pre-treatment. This was also observed with primary patient cells. Mcl-1 levels increased after 6 hours of B exposure, likely due to proteasome inhibition, but decreased by 24 hours with associated cleavage. This effect was concentration-dependent with partial cleavage observed at 4nM (approximately EC30) and full cleavage at 50nM. Bim was present in untreated cells, unchanged after 6 hours of B exposure, but decreased at 24 hours at both concentrations. All of these changes were associated with cleavage of caspase-3 and the appearance of cleaved PARP, and persisted out to 48 hours exposure. Six hours following M exposure, there was an increase in Mcl-1 at the sub-toxic 10uM concentration (possibly a cell survival response), but a reduction at 100uM. Following 24/48 hour exposures changes were no different to control cells with 10uM M, however at 100uM (EC50 concentration) cleavage of Mcl-1 and a decrease in Bim were observed, similar to changes seen with 50nM B. On combining the two drugs simultaneously in a 48 hour exposure, B 4nM and M 10uM failed to induce any changes in U266 cells, but resulted in partial cleavage of Mcl-1 in RPMI 8226 cells. When the M concentration was increased to 100uM there was a decrease of both Mcl-1 and Bim and the associated cleavage of caspase-3 and PARP. There were no differences whether B preceded or followed M. Cell cycle analysis demonstrated G2 arrest following B therapy at 24 hours and in combination with M. Conclusions: This work demonstrates that in multiple myeloma, both Mcl-1 and Bim are closely involved in proteasome mediated cellular apoptosis and in M mediated cytotoxicity. In keeping with work in Jurkat cells (Nencioni et al., Blood 2005), Mcl-1 was found to transiently increase following proteasome inhibition, but then decreased at 24 hours as apoptosis occurred. An early rise in anti-apoptotic proteins such as Mcl-1 may explain why synergistic responses with B and M were seen only with M pretreatment.

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Ibrutinib Sensitizes AML Cells to ROS Inducers Via a BTK-Independent Mechanism

Blood ◽

10.1182/blood.v124.21.2226.2226 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2226-2226 ◽

Cited By ~ 3

Author(s):

Lianne E Rotin ◽

Marcela Gronda ◽

Neil Maclean ◽

Feng-Hsu Lin ◽

Jeff Wrana ◽

...

Keyword(s):

Cell Viability ◽

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Synergistic Effects ◽

Single Agent ◽

B Cell Lymphoma ◽

Fold Increase ◽

Synergistic Cytotoxicity ◽

Btk Inhibitor

Abstract Ibrutinib is a small-molecule Bruton’s tyrosine kinase (BTK) inhibitor yielding impressive clinical responses in B-cell malignancies, where BTK contributes to disease development and progression. We noted that BTK is expressed and constitutively active in acute myeloid leukemia (AML) cell lines and in a subset of AML patients. We therefore sought to determine BTK’s potential role in AML. Using BTK-pY223 expression on immunoblot as a marker of BTK activity, we demonstrated that ibrutinib doses as low as 1μM were sufficient to inhibit BTK activity in the AML cells TEX and OCI-AML2. Yet, these cell lines were insensitive to ibrutinib compared to the B-cell lymphoma Daudi cell line (IC50 10.4μM and 23.7μM versus 1.1μM, respectively). Although inactive as a single agent, we sought to identify drug combinations that sensitized AML cells to pharmacologically relevant concentrations of ibrutinib by conducting a combination chemical screen with our in-house known drug library in TEX and OCI-AML2 cells. According to excess-over-Bliss additivism criteria, we determined that in both cell lines, the poly(ADP-ribose) glycohydrolase inhibitor ethacridine lactate was the most synergistically cytotoxic, producing an 8-fold reduction in the IC50 of ibrutinib. Synergistic cytotoxicity was also observed in a subset of primary AML cells, but not normal hematopoietic cells. Mechanistically, the combination’s synergistic cytotoxicity resulted from excessive ROS production (>10-fold increase, versus a <2-fold increase with either drug alone in TEX and OCI-AML2), since α-tocopherol addition to combination-treated cells rescued cell viability from 24% to 85% and from 4% to 84% in TEX and OCI-AML2, respectively. Combining ibrutinib with other ROS-inducing agents such as parthenolide and the first-line AML therapy daunorubicin produced similar synergistic effects in AML cells,with α-tocopherol also rescuing cell viability. However, the synergistic effects were likely not related to inhibition of BTK, as knockdown of BTK with shRNA did not sensitize TEX cells to ethacridine or daunorubicin and thus did not recapitulate the effects of ibrutinib. We therefore conclude that the BTK inhibitor ibrutinib lacks single agent anti-AML activity, but synergizes with ROS-inducing agents including daunorubicin by inhibiting targets beyond BTK. Thus, clinical trials of ibrutinib in combination with standard chemotherapy could be warranted in AML. Disclosures No relevant conflicts of interest to declare.

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The Effect of Combination of CDK Inhibitor (Roscovitine) and Derivatives of Tetracycline on Human Leukemic Cells.

Blood ◽

10.1182/blood.v108.11.4562.4562 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4562-4562

Author(s):

Hairong Song ◽

Zuzana Hassan

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Cell Lines ◽

Cytotoxic Effect ◽

Single Agent ◽

K562 Cells ◽

Jurkat Cells ◽

Leukemic Cells ◽

Cdk Inhibitor ◽

Derivatives Of

Abstract Roscovitine is a 2,6,9- trisubstituted aminopurine analogue that compete with ATP for binding to the active site of Cyclin-dependent kinases (CDKs). It inhibits CDK2/cyclinE, CDK7/cyclinH and CDK9/cyclinT. The cytotoxic effect of roscovitine and its analogues has been reported in several cancer cell lines in vitro and in animal models of cancer xenografts in vivo. The analogues of tetracycline (Doxycycline and Minocycline) possess anti-tumor, anti-metastatic and anti-angiogenesis properties with low toxicity besides acting as antibiotics. The effect of single agent is usually limited in cancer therapy and the use of single agent may mediate drug resistance. We have studied a cytotoxic effect of the CDK inhibitor roscovitine and tetracycline derivatives either alone or in combinations in human leukemia cells lines HL-60 (myeloid), Jurkat (lymphoblastic) and K562 (CML). HL-60, Jurkat and K562 cells were cultured in RPMI 1640 supplemented with 10% FBS. Cells were treated with roscovitine (0.5 – 100 μM), doxycyline (0.5 – 100 μg/ml) or minocycline (0.5 – 100 μg/ml) up to 48 hours alone or in combinations. The cells were examined for viability using AlarmaBlue assay, proliferation using 3H-thymidine incorporation assay, apoptosis using morphological criteria in Giemsa staining, cell cycle using propidium iodide and flow cytometry. Mitochondrial membrane permeabilization was assessed using tetramethylrhodamine ethyl ester. Specific proteins were detected by Western blotting. Incubation with roscovitine, doxycycline or minocycline decreased the cell viability in concentration-dependent manner in all three cell lines. Pretreatment of cells with doxycycline and minocycline for 24 hours followed by roscovitine for 24 hours have shown the additional decrease in cell viability. No such effect was observed when the drugs were used concomitantly. Roscovitine alone decreased proliferation in concentration- and time-dependent way in all three cell lines. Apoptotic morphology was firstly observed at 3 hrs after the treatment with roscovitine and markedly increased at 6 hrs in HL60 and Jurkat cells, but not in K562 cells. In HL60 and Jurkat cells, the cell cycle analysis has shown an increase in sub-G1 cells at 6 hours with maximum at 24h without preceding cell cycle arrest. In K562 cells sub-G1 peak increased subsequently to G2/M arrest. A marked loss of ΔΨm was observed at 2 hrs of exposure to 25uM of roscovitine in Jurkat and HL-60 cell lines, respectively. In HL60 and Jurkat cells, release of cytochrome c, AIF and Smac from mitochondria into cytosol were detected along with activation of caspases and PARP. However in K562, no caspase activation was detected at studied time points. Thus, all three agents have a cytotoxic effect in different types of leukemic cells, however, the additional cytotoxic effect of combination of Roscovitine and derivatives of tetracycline might be a potent therapeutic approach in treatment of leukemia.

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Combined Inhibition of AKT and KIT Restores Expression of Programmed Cell Death 4 (PDCD4) in Gastrointestinal Stromal Tumor

Cancers ◽

10.3390/cancers13153699 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3699

Author(s):

Marya Kozinova ◽

Shalina Joshi ◽

Shuai Ye ◽

Martin G. Belinsky ◽

Dinara Sharipova ◽

...

Keyword(s):

Cell Death ◽

Gastrointestinal Stromal Tumor ◽

Cell Lines ◽

Synergistic Effects ◽

Single Agent ◽

Xenograft Model ◽

Stromal Tumor ◽

In Vivo Studies ◽

Preclinical Model ◽

Kit Mutation

The majority of gastrointestinal stromal tumor (GIST) patients develop resistance to the first-line KIT inhibitor, imatinib mesylate (IM), through acquisition of secondary mutations in KIT or bypass signaling pathway activation. In addition to KIT, AKT is a relevant target for inhibition, since the PI3K/AKT pathway is crucial for IM-resistant GIST survival. We evaluated the activity of a novel pan-AKT inhibitor, MK-4440 (formerly ARQ 751), as monotherapy and in combination with IM in GIST cell lines and preclinical models with varying IM sensitivities. Dual inhibition of KIT and AKT demonstrated synergistic effects in IM-sensitive and -resistant GIST cell lines. Proteomic analyses revealed upregulation of the tumor suppressor, PDCD4, in combination treated cells. Enhanced PDCD4 expression correlated to increased cell death. In vivo studies revealed superior efficacy of MK-4440/IM combination in an IM-sensitive preclinical model of GIST compared with either single agent. The combination demonstrated limited efficacy in two IM-resistant models, including a GIST patient-derived xenograft model possessing an exon 9 KIT mutation. These studies provide strong rationale for further use of AKT inhibition in combination with IM in primary GIST; however, alternative agents will need to be tested in combination with AKT inhibition in the resistant setting.

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Effect of 5'-fluoro-2'-deoxycytidine and sodium butyrate on the genes of the intrinsic apoptotic pathway, p21, p53, cell viability, and apoptosis in human hepatocellular carcinoma cell lines

Iranian Journal of Pediatric Hematology & Oncology ◽

10.18502/ijpho.v11i4.7163 ◽

2021 ◽

Author(s):

Masumeh Sanaei ◽

Fraidoon Kavoosi ◽

Mohammad Amin Moezzi

Keyword(s):

Gene Expression ◽

Hepatocellular Carcinoma ◽

Cell Viability ◽

Cell Lines ◽

Sodium Butyrate ◽

Cell Apoptosis ◽

Chromatin Organization ◽

Expression Level ◽

Intrinsic Apoptotic Pathway ◽

Apoptotic Pathway

Backgrounds: Epigenetic regulation such as DNA methylation plays a major role in chromatin organization and gene transcription. Additionally, histone modification is an epigenetic regulator of chromatin structure and influences chromatin organization and gene expression. The relationship between DNA methyltransferase (DNMTs) expression and promoter methylation of the tumor suppressor genes (TSGs) has been reported in various cancers. Previously, the effect of 5-aza-2'-deoxycytidine (5-AZA-CdR), trichostatin A (TSA), and valproic acid (VPA) was shown on various cancers. This study aimed to investigate the effect of 5'-fluoro-2'-deoxycytidine (FdCyd) and sodium butyrate on the genes of the intrinsic apoptotic pathway, p21, p53, cell viability, and apoptosis in human hepatocellular carcinoma SNU449, SNU475, and SNU368 cell lines. Materials and Methods: In this lab trial study, the SNU449, SNU475, and SNU368 cells were cultured and treated with 5'-fluoro-2'-deoxycytidine and sodium butyrate. To determine cell viability, cell apoptosis, and the relative gene expression level, MTT assay, flow cytometry assay, and qRT-PCR were done respectively. Results: 5'-fluoro-2'-deoxycytidine and sodium butyrate changed the expression level of the BAX, BAK, APAF1, Bcl-2, Bcl-xL, p21, and p53 gene (P<0.0001) by which induced cell apoptosis and inhibit cell growth in all three cell lines, SNU449, SNU475, and SNU368. Conclusion: Both compounds played their roles through the intrinsic apoptotic pathway to induce cell apoptosis.

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Upregulated JAK/STAT Signaling Represents a Major Mode of Resistance to HDAC Inhibition In Lymphoma and Provides a Rationale for Novel Combination Therapy

Blood ◽

10.1182/blood.v116.21.434.434 ◽

2010 ◽

Vol 116 (21) ◽

pp. 434-434 ◽

Cited By ~ 1

Author(s):

Jason Smith ◽

Katherine J. Walsh ◽

Cassandra L Jacobs ◽

Qingquan Liu ◽

Siyao Fan ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Single Agent ◽

Acquired Resistance ◽

Hematologic Malignancies ◽

Cross Resistance ◽

Clinical Activity ◽

Molecular Pathways ◽

Major Mode ◽

High Degree

Abstract Abstract 434 Background Histone deacetylase inhibitors (HDACis) have demonstrated significant clinical activity in hematologic malignancies; however, single agent response rates have ranged between 20–50% with the duration of response often measured in months, suggesting that drug resistance is a major mode of failure. The pathways through which these agents work and the means by which tumors develop resistance to them are poorly understood. Combination therapy targeting multiple oncogenic pathways holds the promise to improve upon both the depth and durability of these responses. We investigated the mechanisms of inherent and acquired resistance to HDACis in a broad range of lymphomas. By detailing the molecular pathways implicated in resistance to HDACi, we sought to identify novel combinations of compounds that could overcome potential mechanisms that confer resistance. Methods and Results We tested two separate HDACis, LBH589 and SAHA in 51 cell lines representing a wide range of lymphomas including Burkitt lymphoma, diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma, and Hodgkin lymphoma. Gene expression array data was generated for all these cell lines. We then identified genes that were significantly associated with resistance to both LBH589 and SAHA (p<.01) and applied hierarchical clustering to identify the functional significance of these genes. Histology was not predictive of sensitivity to either HDACi. These data were then analyzed using gene set enrichment to identify known molecular pathways associated with resistance. Activation of JAK/STAT signaling was found to be a major determinant of resistance among the cell lines that were relatively resistant to HDACi. (P<0.001, FDR <.25). To determine whether these genes that we found to be associated with resistance reflected potential mechanisms of acquired resistance to HDACi therapy, we separately engineered resistance to LBH589 and SAHA in three DLBCL cell lines (LY3, BJAB, Farage) through incremental dose escalation over a period of up to 6 months. Each of these three cell lines demonstrated sustained growth at drug concentrations that were at or above their original IC50. Each of these cell lines were then exposed to the other HDACi and tested for cross resistance. In each case, the cell lines demonstrated complete cross-resistance to the other drug. We then profiled the gene expression of these cell lines that had acquired resistance. Similar to our previous results, these cell lines demonstrated increased signaling through the JAK/STAT pathway, suggesting that mechanisms of inherent and acquired resistance are similar. We therefore reasoned that combining HDAC and JAK inhibition may overcome both inherent and acquired resistance. To investigate this hypothesis, we tested LBH589 and INCB018424, a JAK1/2 inhibitor, alone and in combination in the LY3, TMD-8, U2932, and BJAB cell lines. While INCB018424 demonstrated no single agent cytotoxicity, it yielded a high degree of synergy when combined with LBH589 with the combination index computed by the Chou-Talalay method ranging from .19 to .9. Conclusion HDACis show single agent activity in the treatment of a number of hematologic malignancies, however most patients develop resistance to these drugs after relatively short-lived remissions. Thus, the greatest promise of these drugs may lie in combination with other agents that target molecular pathways that underlie resistance to these drugs. Using gene expression profiling of a broad range of tumor types and sensitivity to HDACis we were able to identify activation of the JAK/STAT pathway as a common feature of inherent and acquired resistance to HDACis. We combined the JAK1/2 inhibitor INCB018424 with LBH589 and demonstrated a high degree of synergy. As the number of small molecule inhibitors with clinical activity increases, the need to identify rational preclinical combinations becomes greater. Pairing gene expression profiling and resistant cell lines is a promising approach to the selection of combinations likely to maximize clinical benefit while limiting toxicity. Disclosures: No relevant conflicts of interest to declare.

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Exploiting Endogenous Micro-RNAs to Avoid off-Target Transgene Expression

Blood ◽

10.1182/blood.v120.21.3296.3296 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3296-3296

Author(s):

Raul Teruel Montoya ◽

Xianguo Kong ◽

Shaji Abraham ◽

Lin Ma ◽

Leonard C. Edelstein ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Cell Lines ◽

Binding Sites ◽

Transgene Expression ◽

Cell Types ◽

Jurkat Cells ◽

Cell Type ◽

Hematological Disorders

Abstract Abstract 3296 Genetic modification of hematopoietic stem cells (HSCs) has the potential to benefit acquired and congenital hematological disorders. Despite the use of so-called “tissue-specific” promoters to drive expression of the desired transgene, off-target (and consequent deleterious) effects have been observed. MicroRNAs (miRNAs) are important regulators of gene expression. They associate with Argonaute proteins and most typically target 3'UTRs, where complementary base-pairing results in repressed gene expression via RNA decay and translation inhibition. Most miRNAs are ubiquitously expressed, and although some are claimed to be “tissue specific,” such claims have generally not been rigorously validated. The long-term goal of this work is identifying “cell preferential” miRNA expression that could be exploited in expression vectors to minimize off-target transgene expression in HSCs. Initially, total RNA was extracted with Trizol from the megakaryocyte and T-lymphocyte cell lines, Meg-01 and Jurkat, and miRNAs were profiled by Nanostring technology (Nanostring Technologies, Denver, CO). MiR-495 was determined to be highly expressed in Meg-01 and very low in Jurkat cells. A luciferase reporter construct was generated with four canonical binding sites for miR-495 in the 3'UTR and transfected into both cell lines. Compared to control vector without miR-495 binding sites, luciferase expression showed a 50% reduction in Meg-01 cells, but no knock down in Jurkat cells. These experiments indicated that different levels of endogenous miRNA levels can regulate transgene expression through a novel design in the 3'UTR. We next turned our attention to human hematopoietic cells. We reasoned that the long-term goal of minimal off-target transgene expression in HSCs would require knowledge of miRNAs that had little or no detectable expression (“selectively reduced [SR]”) in one cell type and were highly expressed in other cell types. In this manner, the transgene expression would be dampened only in the non-target cells. As a surrogate for bone marrow progenitors and as proof of principle, we used primary cells in normal human peripheral blood. T-cells, B-cells, platelets and granulocytes were purified by density centrifugation followed by immunoselection from five healthy human donors. Flow cytometry using membrane specific markers demonstrate >97% purity of each specific cell preparation. Total RNA was extracted and miRNAs were profiled as above. First, we identified 277 miRNAs that were differentially expressed between any pair of cell types (p-value<0.05 by ANOVA). Second, we performed ranked pair-wise comparisons across all cell types to determine SR miRNAs. This analysis revealed 5 platelet SR-miRNAs, 6 B-cell SR-miRNAs, 2 T-cell SR-miRNAs and 4 granulocyte SR-miRNAs. Lastly, we considered which of these 17 SR-miRNAs would be the best single SR-miRNA within and across cell types. SR-miRNAs were normalized to let-7b, a miRNA we determined to be equivalently expressed across all cell types, and hence, an ideal normalizer. Lineage-specific SR-miRNAs were selected based on extremely low expression in only one cell type and highest fold change of expression compared to the other cell types. The best SR-miRNAs were miR-29b (SR in platelets), miR-125a-5p (SR in B-cells) and miR-146a (SR in granulocytes). The SR expression levels of these 3 miRNAs were validated by qRT-PCR. Our analysis identified no good SR-miRNAs in T-cells. On-going experiments are testing the selective effects of the SR miRNAs in lentiviral vector infection of cord blood CD34+ cells differentiated along specific lineages. In summary, we have demonstrated in hematopoietic cell lines that SR endogenous miRNAs can regulate the expression of transgenes via tandem arrangement of their target sites in the 3'UTR. Additionally, we have identified miRNAs that are specifically expressed at a very low level in one blood cell type and at high levels in other cell types. These miRNAs could potentially be utilized as new biological tools in gene therapy for hematological disorders to restrict transgene expression and avoid the negative consequences of off-target expression. Disclosures: No relevant conflicts of interest to declare.

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Anti-Myeloma Effects of the Janus Kinase 2 (JAK2) Inhibitor SAR503 Alone and in Combination Treatment

Blood ◽

10.1182/blood.v120.21.4986.4986 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4986-4986

Author(s):

Haiming Chen ◽

Mingjie Li ◽

Jennifer Li ◽

Kevin Delijani ◽

Danielle Rauch ◽

...

Keyword(s):

Cell Proliferation ◽

Tumor Cell ◽

Cell Viability ◽

Tumor Cells ◽

Cell Lines ◽

Drug Exposure ◽

Single Agent ◽

Janus Kinase ◽

Gm Csf ◽

Jak2 Inhibitor

Abstract Abstract 4986 Background: Janus kinase 2 (JAK2) is a cytoplasmic tyrosine kinase that carries out a series of cascading signals via signal transducer and activator of transcription (STAT)s, mitogen-activated protein kinase (MAPK), and phosphorylation of PI3K. Activation of the JAK2 pathway plays an important role in both normal and malignant hematopoiesis. The JAK pathway ha been shown to play a key role in multiple myeloma (MM). JAK2 has been specifically implicated in signaling by members of the type II cytokine receptor family (interferon [IFN] receptor), GM-CSF receptor (IL-3R, IL-5R, and GM-CSF-R), gp130 receptor family interleukin-6 (IL-6R) and single chain receptors (Epo-R, Tpo-R, GH-R, and PRL-R). IFN-α inhibits MM cell proliferation in association with cell cycle arrest at G1 and limits the clonogenic growth of both MM cell lines and primary MM patient specimens. SAR503 (Sanofi-Aventis) is a potent, highly selective JAK2 inhibitor. Thus, we evaluated the anti-MM effects of SAR503 as a single agent and in combination with other anti-MM drugs and evaluated gene and protein expression in MM cells exposed to these drugs. Experiment design: The MM cell lines RPMI8226, U266, and MM1s were cultured in RPMI1640 with standard nutrition supplements. Bone marrow aspirates were obtained from MM patients following informed consent. Bone marrow mononuclear cells (BMMCs) were isolated by using density-gradient centrifugation with Histopaque-1077 (Sigma, St Louis). Cells were plated in 96 well plates at a concentration of 6 × 104 cells/100 ml/well, and incubated for 24 hours prior to drug treatment, after which time the drugs were added in replicates of six for 48 hours. BMMCs were incubated in the presence of media, SAR503, doxorubicin, melphalan, dexamethasone, bortezomib, or IFN-α alone or the combination of SAR503 with one of these anti-MM agents. Following the 48-hour drug incubation, cell viability was assessed utilizing the cell proliferation MTS assay. For gene expression studies, total RNA was isolated MM tumor cells with or without drug exposure. RNA was reverse-transcribed into cDNA and amplified using the Thermo-Script RT-PCR System and PCR performed again using the GeneAmp PCR System 9700. Protein phosphorylation of MM tumor cells with or without drug exposure was determined with Western blot analysis. Results: SAR503 alone inhibited MM tumor cell proliferation in a concentration-dependent fashion. The 50% growth inhibition (IC50) of cells from MM cell lines at 48 hours varied (IC50: RPMI8226 1mM; U266 0. 5mM; MM1s 10mM). IC50 of primary MM tumor cells treated with SAR503 ranged from approximately 5 to 10mM in different patients. Notably, the combination of SAR503 and either doxorubicin or melphalan showed markedly reduced cell viability compared to either drug alone in all three MM cell lines and primary tumor cells from MM patients. Since this effect may have resulted from decreased cell proliferation due to inhibition of the JAK2 pathway and cell cycle arrest or increased cell death, we further determined cell apoptosis of MM tumor cells treated with SAR503 alone by using flow cytometric analysis to detect Annexin V and propidium iodide (PI) staining. Our data showed SAR503 increased MM tumor cell apoptosis in a concentration-dependent fashion. The combination of SAR503 and dexamethasone or bortezomib only slightly reduced tumor cell viability in both MM cell lines and primary MM tumor cells more than single agent treatment, and the combination of SAR503 with IFN-α did not enhance the anti-MM effects compared to single drug treatment. Notably, RT-PCR results showed marked decreases in both AKT1 and mTOR gene expression in MM tumor cells treated with SAR503. Conclusion: The combination of the JAK2 inhibitor SAR503 with doxorubicin or melphalan markedly reduces MM tumor cell viability more than single agent treatment. The results from these studies suggest that enhanced anti-MM activity may be observed when SAR503 is combined with conventional treatment for MM. We are currently evaluating the anti-MM effects of SAR503 in these combination treatments in vivo using our MM xenograft models. Disclosures: Berenson: Onyx: Consultancy, Honoraria, Speakers Bureau.

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Deletions In TBL1XR1 Results In Glucocorticoid Resistance By Decreasing Glucocorticoid Signaling In Childhood B-Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v122.21.602.602 ◽

2013 ◽

Vol 122 (21) ◽

pp. 602-602

Author(s):

Courtney L Jones ◽

Teena Bhatla ◽

Jinhua Wang ◽

Wallace Bourgeois ◽

Bitterman S Danielle ◽

...

Keyword(s):

Gene Expression ◽

Cell Viability ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Annexin V ◽

Steroid Resistance ◽

Rt Pcr ◽

Regulatory Regions ◽

Prednisolone Treatment ◽

Gene Regulatory

Abstract Introduction The prognosis for children with acute lymphoblastic leukemia who relapse is poor and discovery of the underlying mechanisms that lead to drug resistance is a top priority. Relapsed blasts have intrinsic chemoresistance compared to diagnosis blasts especially to glucocorticoids (Klumper et. al, 1995). Furthermore, resistance to glucocorticoids is associated with a poor prognosis in childhood ALL (Dördelmann M et. al, Blood 1999, Schmiegelow K et. al, Leukemia 2001, Tissing WJ et. al, Leukemia 2003). We have previously identified recurrent deletions with concordant decreased gene expression in TBL1XR1 in 10.7% of patients at relapse (Hogan et. al, 2011). TBL1XR1 codes for the TBLR1 protein which is responsible for the dismissal and degradation of nuclear corepressor (N-CoR) complex proteins including N-CoR1, SMRT, GPS2, and histone deacetylases (HDAC) (Perissi V et. al, Cell 2004). We hypothesized that TBL1XR1 deletions may result in resistance to glucocorticoid agonist, prednisolone through up-regulation of N-CoR complex proteins. Methods B-precursor ALL cell lines Reh, and RS4;11 were transduced with lentiviral constructs containing control and TBL1XR1 targeting shRNAs. Knockdown was confirmed by RT-PCR and western blotting. Stable cell lines were treated with prednisolone, doxorubicin, 6-thioguanine, or etoposide for 24-48 hours. Cell viability and apoptosis were measured by cell titer glo luminescence assay (promega) and annexin V-PE and 7-Amino-actinomycin D (7AAD) staining (Annexin V-PE Apoptosis Detection Kit, BD Pharmingen, San Diego, CA, USA) respectively. To determine changes in global gene expression by TBL1XR1 knockdown, stable Reh cell lines were treated with prednisolone or vehicle for 8 hours and then collected for RNA extraction (Qiagen, RNeasy mini kit) and microarray analysis. Microarray data was validated by RT-PCR. To elucidate the mechanism of resistance we performed small-scale biochemical fractionation and chromatin immunoprecipitation (ChIP) detecting levels of glucocorticoid receptor (GR), TBLR1, N-CoR1, and HDAC3 residing on the chromatin as well as gene specific glucocorticoid response elements (GREs). Results In this study, we demonstrate that knockdown of TBL1XR1 results in resistance to the glucocorticoid agonist prednisolone but not other classes of chemotherapeutic agents. We discovered that 51 of the 117 genes induced by prednisolone in control cells had decreased induction of at least 50%. We validated a subset of prednisolone induced genes including, GILZ, TXNIP, ZEB1, ST6GALNAC3, IL21R, and CCPG1 by RT-PCR. To explore the mechanism of TBL1XR1 mediated decrease in GR signaling we determined the effect of TBL1XR1 depletion of GR recruitment to total bulk chromatin. In TBL1XR1 knockdown cells, no GR was detected in the chromatin associated fractions in vehicle or prednisolone treatment conditions, despite similar levels of GR protein between control and TBL1XR1 knock down lines. We show that the decreased GR levels is associated with an increased level of NCoR1 detected in the chromatin fraction of TBLR1 depleted cells; however no change in HDAC3 levels were observed. We confirmed these results by interrogating the gene regulatory regions of GILZ and TXNIP by ChIP. In TBL1XR1 depleted lines a decrease in GR occupancy in prednisolone stimulated cells was observed compared to control lines. We also observed increased levels of N-CoR1, and HDAC3 occupying these GREs. To interrogate the functional relationship between increased NCoR1 and HDAC3 levels on the gene regulatory region as a result of TBL1XR1 knockdown we depleted NCoR1 or inhibited HDAC3 using a pan HDAC inhibitor SAHA and examined the impact of prednisolone treatment on cell viability and induction of GILZ. We found that upon NCoR1 depletion or HDAC inhibition, TBL1XR1 knockdown line was no longer resistant to prednisolone and the induction of GILZ was restored. Conclusions Reduction of TBL1XR1 results in prednisolone resistance in ALL by decreasing GR occupancy on gene regulatory regions through the upregulation of the NCoR co-repressor complex at these sites. Our work and others has provided insight into the importance of transcription regulatory complexes in steroid resistance in ALL (and perhaps other malignancies) as well as opportunities for novel therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

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