Chemical Genomics Reveals JAK STAT Activation As a Mechanism of Resistance to HDAC Inhibitors in B Cell Lymphomas

Abstract Background: HDAC inhibitors (HDACi) are being investigated as treatment for relapsed/refractory non Hodgkin lymphoma (NHL) and other cancers. However, the mechanisms underlying sensitivity and resistance to HDAC inhibition in lymphomas have not been fully characterized. We probed the cellular and molecular response to HDACi in vitro and in vivo in order to determine factors that dictate the response to HDACi and to enable design of approaches to incorporate HDACi into novel combination therapeutics. Methods: High-throughput cytotoxicity screening was performed using two different HDAC inhibitors, LBH589 (panobinostat) and SAHA (vorinostat) in 52 lymphoid cell lines characterized through RNA-seq and microarray gene expression profiling. This screen revealed a greater than 50-fold range in concentration needed to induce cytotoxicity for the 2 different HDAC inhibitors and there was moderate correlation between the 2 compounds in this panel (Pearson correlation r = 0.76, p < 0.01). By pairing this chemosensitivity data with gene expression profiles of the screened cell lines, we developed a gene expression classifier for LBH589 that identified resistant and sensitive cell line groups. This predictor was applied to B-cell NHL cell lines tested with LBH589 in the Cancer Cell Line Encyclopedia (CCLE) and we found that the sensitive and resistant cell line groups distinguished by this method differed more than 5-fold in IC50 (0.021 vs. 1.24 nM, P < 0.01 by Wilcoxon rank sum), thus validating the ability of this approach to distinguish HDACi resistant cell lines. We further initiated a clinical trial of LBH589 in relapsed/refractory diffuse large B cell lymphoma patients combined with RNAseq profiling of their tumors prior to embarking on treatment. We treated nine patients with LBH589, and application of our response predictor to scaled RNAseq gene expression data revealed 4 predicted responders and 5 predicted non-responders. Two of the predicted responders had a clinical response to LBH589, whereas none of the predicted non-responders had a clinical response, thus our classifier was able to identify all of the LBH589-responsive patients from this cohort (P = 0.08 by Fisher's exact test). Analysis of differentially expressed molecular pathways in HDACi sensitive and resistant samples by gene set enrichment revealed the JAK-STAT pathway as the most differentially expressed pathway associated with HDACi resistance (at P < 0.001 and FDR < 0.20). We further identified a number of distinct mutations including STAT3, SOCS1 and JAK1 that were associated with activation of the JAK-STAT pathway by gene expression signatures and the LBH589 response signature in DLBCL cell lines and patient samples by analysis of RNA-seq data. Phosphoprotein analysis by Western blot and Sis-inducible-element (SIE) luciferase reporter assays were used to confirm JAK-STAT activation in these samples and we found that overexpression of STAT3 Src-homology domain mutations activated JAK-STAT3 signaling in isogenic cell lines and fostered resistance to LBH589 in vitro. Conversely, using in vivo DLBCL xenograft models, we found that combining JAK-STAT and HDAC inhibition by treatment with LBH589 and ruxolitinib resulted in synergistic reduction of tumor cell viability and tumor growth with tolerable toxicity in mice. Conclusions: Sustained JAK-STAT activation appears to mediate resistance to HDAC inhibition in DLBCL and other NHLs and several recurrent genetic lesions drive JAK-STAT activation in these diseases. This process can be overcome by JAK 1/2 inhibition with ruxolitinib and these findings demonstrate a role for combination therapy with HDAC inhibitors and small molecules targeting the JAK-STAT pathway in lymphoid malignancies. Disclosures No relevant conflicts of interest to declare.

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Histone Deacetylase Inhibition Using LBH589 Is Effective in Lymphoma and Results in Down-Regulation of the NF-KB Pathway.

Blood ◽

10.1182/blood.v114.22.3730.3730 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3730-3730 ◽

Cited By ~ 2

Author(s):

Jason L. Smith ◽

Amee Patel ◽

Siyao Fan ◽

Cassandra L. Jacobs ◽

Katherine J. Walsh ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Cell Lines ◽

Expression Profiles ◽

Hdac Inhibitors ◽

Gene Expression Profiles ◽

Resistant Cell ◽

Hdac Inhibition ◽

Down Regulation ◽

Highly Sensitive

Abstract Abstract 3730 Poster Board III-666 Background Histone deacetylase (HDAC) inhibition has emerged as a promising therapeutic approach in malignancies. HDAC inhibition has proved to be a particularly effective option in patients with lymphoma. The HDAC inhibitor vorinostat is approved for the treatment of patients with cutaneous T-cell lymphomas and is being tested in patients with B cell lymphomas. More recently, a number of other HDAC inhibitors have entered preclinical and clinical testing. The mechanisms through which HDAC inhibitors exert their downstream effects are currently unknown. As the number of HDAC inhibitors in development increases, it is unclear if they share a class effect or display unique mechanisms of action. Recently, LBH589 has been described as an orally available, highly potent inhibitor of HDAC. We decided to explore whether LBH589 would be an effective therapeutic option for patients with lymphoma. Methods and Results In order to evaluate whether LBH was efficacious and potent in B cell lymphomas, we tested both vorinostat and LBH589 in the same cell line(s). We found that LBH589 was over 10 times more potent than vorinostat (mean IC50 7.4nM versus 830nM). We decided to further test LBH589 in an expanded panel of 18 cell lines derived from 5 different lymphoid malignancies: Burkitt lymphoma, mantle cell lymphoma, Hodgkin lymphoma, multiple myeloma and diffuse large B cell lymphoma. LBH589 was found to be lethal in each of these cell lines at IC50 concentrations varying from 5.6-31.5 nM (mean 11.2nM), suggesting that this drug may be effective at physiologically achievable concentrations. Based on the IC50 cut-off of 10nM, we assigned the treated cell lines to 2 groups: highly sensitive (IC50 < 10nM, N=11) and less sensitive (IC50> 10nM, N=8). We performed gene expression profiling on 12 of these cell lines and compared the gene expression profiles of the highly sensitive versus less sensitive cell lines. Further, we performed time course experiments in which we evaluated the effects of LBH589 at its IC50 on cell lines at 6 and 12 hours post-treatment. Gene expression profiling was performed on the treated cells at each time point. We also engineered resistant cell lines by incremental dose escalation over a period of months to a concentration greater than or equal to the IC50. The resistant cell lines were also profiled for gene expression and compared to the wild type cell lines. The gene expression profiles of LBH589 treated cells at 6 and 12 hours demonstrated a clear and progressive down regulation of genes associated with the NF-KB pathway (Figure 1). Furthermore, cell lines with high expression of genes in the NF-KB pathway were uniformly highly sensitive to LBH589 with IC50<10nM in all cases. Conclusion NF-KB activation is a common feature of many different lymphoma types. Our data suggest that HDAC inhibition using LBH589 could provide a potent method for treating lymphomas and that HDAC inhibitors may exert their effects through the down-regulation of the NF-KB pathway. Our data also suggest a rationale for dual inhibition of HDAC and NF-KB in the treatment of lymphoma. Disclosures: Rizzieri: Merck & Co., Inc.: Consultancy.

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Establishment of B-Cell Lymphoma Cell Lines Persistently Infected with Hepatitis C Virus In Vivo and In Vitro: the Apoptotic Effects of Virus Infection

Journal of Virology ◽

10.1128/jvi.77.3.2134-2146.2003 ◽

2003 ◽

Vol 77 (3) ◽

pp. 2134-2146 ◽

Cited By ~ 195

Author(s):

Vicky M.-H. Sung ◽

Shigetaka Shimodaira ◽

Alison L. Doughty ◽

Gaston R. Picchio ◽

Huong Can ◽

...

Keyword(s):

Hepatitis C ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Culture System ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Hcv Rna

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkin's B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells (PBMCs), and an established B-cell line (Raji cells) in vitro. The virus from SB cells belongs to genotype 2b. Single-stranded conformational polymorphism and sequence analysis of the viral RNA quasispecies indicated that the virus present in SB cells most likely originated from the patient's spleen and had an HCV RNA quasispecies pattern distinct from that in the serum. The virus production from the infected primary hepatocytes showed cyclic variations. In addition, we have succeeded in establishing several Epstein-Barr virus-immortalized B-cell lines from PBMCs of HCV-positive patients. Two of these cell lines are positive for HCV RNA as detected by reverse transcriptase PCR and for the nonstructural protein NS3 by immunofluorescence staining. These observations unequivocally establish that HCV infects B cells in vivo and in vitro. HCV-infected cell lines show significantly enhanced apoptosis. These B-cell lines provide a reproducible cell culture system for studying the complete replication cycle and biology of HCV infections.

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Targeted Inhibition of PI3K α/δ Is Synergistic with BCL-2 Blockade in Genetically Defined Subtypes of DLBCL

Blood ◽

10.1182/blood-2018-99-113647 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 39-39

Author(s):

Kamil Bojarczuk ◽

Kirsty Wienand ◽

Jeremy A. Ryan ◽

Linfeng Chen ◽

Mariana Villalobos-Ortiz ◽

...

Keyword(s):

Tumor Growth ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Research Funding ◽

Single Agent ◽

Research Report ◽

Genetic Bases

Abstract Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease that is transcriptionally classified into germinal center B-cell (GCB) and activated B-cell (ABC) subtypes. A subset of both GCB- and ABC-DLBCLs are dependent on B-cell receptor (BCR) signaling. Previously, we defined distinct BCR/PI3K-mediated survival pathways and subtype-specific apoptotic mechanisms in BCR-dependent DLBCLs (Cancer Cell 2013 23:826). In BCR-dependent DLBCLs with low baseline NF-κB activity (GCB tumors), targeted inhibition or genetic depletion of BCR/PI3K pathway components induced expression of the pro-apoptotic HRK protein. In BCR-dependent DLBCLs with high NF-κB activity (ABC tumors), BCR/PI3K inhibition decreased expression of the anti-apoptotic NF-κB target gene, BFL1. Our recent analyses revealed genetic bases for perturbed BCR/PI3K signaling and defined poor prognosis DLBCL subsets with discrete BCR/PI3K/TLR pathway alterations (Nat Med 2018 24:679). Cluster 3 DLBCLs (largely GCB tumors) exhibited frequent PTEN deletions/mutations and GNA13 mutations. Cluster 5 DLBCLs (largely ABC tumors) had frequent MYD88L265P and CD79B mutations that often occurred together. These DLBCL subtypes also had different genetic mechanisms for deregulated BCL2 expression - BCL2 translocations in Cluster 3 and focal (18q21.33) or arm level (18q) BCL2 copy number gains in Cluster 5. These observations prompted us to explore the activity of PI3K inhibitors and BCL2 blockade in genetically defined DLBCLs. We utilized a panel of 10 well characterized DLBCL cell line models, a subset of which exhibited hallmark genetic features of Cluster 3 and Cluster 5. We first evaluated the cytotoxic activity of isoform-specific, dual PI3Kα/δ and pan-PI3K inhibitors. In in vitro assays, the PI3Kα/δ inhibitor, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs. We next assessed the transcriptional abundance of BCL2 family genes in the DLBCLs following copanlisib treatment. In BCR-dependent GCB-DLBCLs, there was highly significant induction of the pro-apoptotic HRK. In BCR-dependent ABC-DLBCLs, we observed significant down-regulation of the anti-apoptotic BFL1 protein and another NF-κB target gene, BCLxL (the anti-apoptotic partner of HRK). We then used BH3 profiling, to identify dependencies on certain BCL2 family members and to correlate these data with sensitivity to copanlisib. BCLxL dependency significantly correlated with sensitivity to copanlisib. Importantly, the BCLxL dependency was highest in DLBCL cell lines that exhibited either transcriptional up-regulation of HRK or down-regulation of BCLxL following copanlisib treatment. In all our DLBCL cell lines, PI3Kα/δ inhibition did not alter BCL2 expression. Given the genetic bases for BCL-2 deregulation in a subset of these DLBCLs, we next assessed the activity of the single-agent BCL2 inhibitor, venetoclax, in in vitro cytotoxicity assays. A subset of DLBCL cell lines was partially or completely resistant to venetoclax despite having genetic alterations of BCL2. We postulated that BCR-dependent DLBCLs with structural alterations of BCL2 might exhibit increased sensitivity to combined inhibition of PI3Kα/δ and BCL2 and assessed the cytotoxic activity of copanlisib (0-250 nM) and venetoclax (0-250 nM) in the DLBCL cell line panel. The copanlisib/venetoclax combination was highly synergistic (Chou-Talalay CI<1) in BCR-dependent DLBCL cell lines with genetic bases of BCL2 deregulation. We next assessed copanlisib and venetoclax activity in an in vivo xenograft model using a DLBCL cell line with PTENdel and BCL2 translocation (LY1). In this model, single-agent copanlisib did not delay tumor growth or improve survival. Single-agent venetoclax delayed tumor growth and improved median survival (27 vs 51 days, p<0.0001). Most notably, we found that the combination of copanlisib and venetoclax delayed tumor growth significantly longer than single-agent venetoclax (p<0.0001). Additionally, the combined therapy significantly increased survival in comparison with venetoclax alone (median survival 51 days vs not reached, p<0.0013). Taken together, these results provide in vitro and in vivo pre-clinical evidence for the rational combination of PI3Kα/δ and BCL2 blockade and set the stage for clinical evaluation of copanlisib/venetoclax therapy in patients with genetically defined relapsed/refractory DLBCL. Disclosures Letai: AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Novartis: Consultancy, Other: Lab research report; Vivid Biosciences: Equity Ownership; AstraZeneca: Consultancy, Other: Lab research report. Shipp:AstraZeneca: Honoraria; Merck: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding.

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Pharmacologic Inhibition of the Histone Methyltransferase SETD2 with EZM0414 As a Novel Therapeutic Strategy in Relapsed or Refractory Multiple Myeloma and Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood-2021-151147 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1142-1142

Author(s):

Jennifer Totman ◽

Dorothy Brach ◽

Vinny Motwani ◽

Selene Howe ◽

Emily Deutschman ◽

...

Keyword(s):

Multiple Myeloma ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

B Cell Lymphoma ◽

Publicly Traded ◽

The Past ◽

Molecule Inhibitor

Abstract Introduction: SETD2 is the only known histone methyltransferase (HMT) capable of catalyzing H3K36 trimethylation (H3K36me3) in vivo. It plays an important role in several biological processes including B cell development and maturation, leading to the hypothesis that SETD2 inhibition in these settings could provide anti-tumor effects. The normal process of B cell development/maturation renders B cells susceptible to genetic vulnerabilities that can result in a dysregulated epigenome and tumorigenesis, including in multiple myeloma (MM) and diffuse large B-cell lymphoma (DLBCL). For example, 15%-20% of MM harbors the high risk (4;14) chromosomal translocation, resulting in high expression of the multiple myeloma SET domain (MMSET) gene. MMSET is an HMT that catalyzes H3K36me1 and H3K36me2 formation and extensive scientific work has established overexpressed MMSET as a key factor in t(4;14) myeloma pathogenesis. To the best of our knowledge MMSET has eluded drug discovery efforts, however, since t(4;14) results in high levels of the H3K36me2 substrate for SETD2, inhibiting SETD2 offers promise for targeting the underlying oncogenic mechanism driven by MMSET overexpression in t(4;14) MM patients. In addition, SETD2 loss of function mutations described to date in leukemia and DLBCL are always heterozygous, suggesting a haploinsufficient tumor suppressor role for SETD2. This observation points to a key role for SETD2 in leukemia and lymphoma biology and suggests that therapeutic potential of SETD2 inhibition may also exist in these or similar settings. EZM0414 is a first-in-class, potent, selective, orally bioavailable small molecule inhibitor of the enzymatic activity of SETD2. We explored the anti-tumor effects of SETD2 inhibition with EZM0414 in MM and DLBCL preclinical studies to validate its potential as a therapy in these tumor types. Methods: Cellular proliferation assays determined IC 50 values of EZM0414 in MM and DLBCL cell line panels. Cell line-derived xenograft preclinical models of MM and DLBCL were evaluated for tumor growth inhibition (TGI) in response to EZM0414. H3K36me3 levels were determined by western blot analysis to evaluate target engagement. Combinatorial potential of SETD2 inhibition with MM and DLBCL standard of care (SOC) agents was evaluated in 7-day cotreatment in vitro cellular assays. Results: Inhibition of SETD2 by EZM0414 results in potent anti-proliferative effects in a panel of MM and DLBCL cell lines. EZM0414 inhibited proliferation in both t(4;14) and non-t(4;14) MM cell lines, with higher anti-proliferative activity generally observed in the t(4;14) subset of MM cell lines. The median IC 50value for EZM0414 in t(4;14) cell lines was 0.24 μM as compared to 1.2 μM for non-t(4;14) MM cell lines. Additionally, inhibitory growth effects on DLBCL cell lines demonstrated a wide range of sensitivity with IC 50 values from 0.023 μM to >10 μM. EZM0414 resulted in statistically significant potent antitumor activity compared to the vehicle control in three MM and four DLBCL cell line-derived xenograft models. In the t(4;14) MM cell line-derived xenograft model, KMS-11, robust tumor growth regressions were observed at the top two doses with maximal TGI of 95%. In addition, two non-t(4;14) MM (RPMI-8226, MM.1S) and two DLBCL xenograft models (TMD8, KARPAS422) demonstrated > 75% TGI; with two additional DLBCL models (WSU-DLCL2, SU-DHL-10) exhibiting > 50% TGI in response to EZM0414. In all models tested, the antitumor effects observed correlated with reductions in intratumoral H3K36me3 levels demonstrating on-target inhibition of SETD2 methyltransferase activity in vivo. In vitro synergistic antiproliferative activity was also observed when EZM0414 was combined with certain SOC agents for MM and DLBCL. Conclusions: Targeting SETD2 with a small molecule inhibitor results in significantly reduced growth of t(4;14) MM, as well as non-t(4;14) MM and DLBCL cell lines, in both in vitro and in vivo preclinical studies. In addition, in vitro synergy was observed with EZM0414 and certain SOC agents commonly used in MM and DLBCL, supporting the combination of SETD2 inhibition with current MM and DLBCL therapies. This work provides the rationale for targeting SETD2 in B cell malignancies such as MM, especially t(4;14) MM, as well as DLBCL, and forms the basis for conducting Phase 1/1b clinical studies to evaluate the safety and activity of EZM0414 in patients with R/R MM and DLBCL. Disclosures Totman: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Brach: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Motwani: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Howe: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Deutschman: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Lampe: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Riera: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Tang: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Eckley: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Alford: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Duncan: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Farrow: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Dransfield: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Raimondi: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Thomeius: Foghorn Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cosmopoulos: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Kutok: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company.

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GSK1838705a, an IGF-1R/ALK Inhibitor, Overcomes Resistance to Crizotinib in ALK-Positive ALCL

Blood ◽

10.1182/blood-2019-130063 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4069-4069

Author(s):

Wenyu Shi ◽

Jian-Yong Li

Keyword(s):

Cell Line ◽

Cell Lines ◽

Large Cell ◽

Resistant Cell ◽

Resistant Cell Line ◽

Expression Level ◽

Downstream Signaling ◽

Single Drug

Anaplastic large cell lymphoma (ALCL) is a type of CD30-expressing non-Hodgkin's lymphoma (NHL), which accounts for 2% to 3% of adult non-Hodgkin's lymphoma,accounting for 15% to 30% of children with large cell lymphoma. Anaplastic lymphoma kinase (ALK) positive ALCL is highly invasive, and currently it is generally based on CHOP combined with chemotherapy. The proportion of patients with complete relief of symptoms is as high as 90%, but the proportion of recurrence is also as high as 40%. Crizotinib is the first generation of ALK inhibitors that have been approved for the treatment of ALK+ ALCL. Unfortunately, most patients treated with crizotinib relapse after a significant initial response. The median progression-free survival of clinical trials was 10.5 months. Various mutations in the ALK kinase domain and amplification of the ALK gene copy number, activation of the alternative pathway, and tumor heterogeneity are major causes of crizotinib resistance. Studies have shown that IGF-1R interacts with NPM-ALK to promote ALK+ALCL transformation, proliferation and migration. GSK is a small molecule kinase inhibitor that inhibits both IGF-IR and ALK. Therefore, GSK with simultaneous inhibition of the bidirectional potential of IGF-IR and ALK has a promising prospect in the targeted therapy of NPM-ALK+ALCL. This study explored the inhibitory effects of GSK on NPM-ALK+ALCL and crizotinib-resistant NPM-ALK+ALCL by in vivo and in vitro experiments. In vitro experiments: The sensitivity of ALCL cell line to GSK1838705a was detected by CCK8 and flow cytometry. The expression of phosphorylation of IGF-1R and NPM-ALK signaling pathway in Karpas299 and SR786 cell lines stimulated by GSK was detected by WB method. In order to study the crizotinib resistance mutation, we established ALK+ALCL crizotinib-resistant cell lines Karpas299-R and SR786-R, and identified the resistance of Karpas299-R and SR786-R cell lines by CCK8 and flow cytometry. The drug-resistant and non-resistant strains were stimulated with gradient concentrations of crizotinib and gradient GSK, and the IC50 of the two were compared by CCK8. The WB method was used to compare the phosphorylation levels of downstream signaling pathways in drug-resistant and non-resistant strains. In vivo experiment: The ALK+ALCL and resistant-ALK+ALCL mouse model was established, and three groups of mice treated with control, GSK single drug 30 mg/kg, GSK single drug 60 mg/kg, were established. The tumor volume and body weight of the four groups were compared. Immunohistochemistry was used to compare the expression levels of key signaling molecules and apoptotic proteins in each group. SPSS statistical software draws survival curves. As the concentration of GSK gradually increases, the survival rate of ALCL cells gradually decreases. The expression of pIGF-1R, pNPM-ALK, pSTAT3, pAKT, casepase3 and other molecules decreased in the downstream signaling pathway, and the expression level of cleaved-casepase3 increased.In the crizotinib-resistant cell line, with the increase of the concentration of GSK, the apoptosis rate of the cells increased and the phosphorylation level of the downstream molecules gradually decreased. Tumor volume of three groups of mouse models: control>GSK single drug 30 mg/kg>GSK single drug 60 mg/kg. Immunohistochemistry results showed that the expression level of key signaling molecules in GSK-treated CHOP-treated mice decreased, and the expression level of apoptotic proteins increased. In this research, we explored the effects of GSK1838705A on proliferation, apoptosis, and clonogenesis of ALCL cell lines. Subsequently, we established a crizotinib-resistant cell line and noticed that GSK1838705A can effectively reduce the viability of resistant ALCL cells and significantly restrain the transmission of downstream survival signaling pathways induced by IGF1R/IR phosphorylation. Besides, we discovered that GSK1838705A inhibited the development of both crizotinib-sensitive and crizotinib-resistant ALCL tumors in the ALCL mouse model established by subcutaneous tumorigenesis. Based on the results of previous clinical trials, we put forward to use GSK1838705A as an alternative treatment strategy to overcome crizotinib-resistant ALCL. Disclosures No relevant conflicts of interest to declare.

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Novel Mechanisms for Resistance to Targeted Therapy Identified through Machine Learning Approaches in 1167 RNA-Seq Drug Exposure Profiles in Lymphoma

Blood ◽

10.1182/blood-2018-99-119639 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1370-1370

Author(s):

Nicholas Davis ◽

Matthew S. McKinney ◽

Anupama Reddy ◽

Cassandra Love ◽

Eileen Smith ◽

...

Keyword(s):

Machine Learning ◽

B Cell ◽

Cell Lines ◽

Learning Approaches ◽

Mechanisms Of Resistance ◽

Pathway Activation ◽

Large B Cell ◽

Stat Pathway

Abstract Introduction: Diffuse large B cell lymphoma (DLBCL) is a clinically heterogeneous disease. While roughly half of the patients respond well to standard R-CHOP therapy, the majority of the remainder succumb to their disease. While many targeted therapies have been developed in DLBCL, resistance to single agents develops almost invariably. While drug combinations have proved to be effective approaches to overcoming resistance in infectious diseases, developing such combinations has proved to be difficult in diffuse large B cell lymphomas and other cancers owing to not only the heterogeneity of these diseases and overlapping toxicity profiles. We hypothesized that with advent of powerful new machine learning approaches combined with genomics, that we would be able to identify novel mechanisms of resistance and develop effective combination therapies to overcome resistance to single agents. Results We tested in vitro responses to all FDA-approved and Phase III cancer drugs (N=150) in six DLBCL cell lines carefully chosen to represent the heterogeneity with regard to cell of origin and common genetic alterations. We observed that roughly half of our drugs were active in at least 50% of the DLBCL cell lines. We then performed RNA sequencing on these cell lines before and after exposure to each of these drugs at their specific IC50 (concentration of drug required to kill 50% of the cells). In addition, we tested the effects of 38 cytokines and antibodies to assess their downstream biological effects. In all, we generated 1167 RNAseq profiles post-exposure to drug (N=900) or cytokines. Hierarchical clustering of our RNAseq data demonstrated clusters of drugs with shared mechanisms and targets (e.g. HDAC inhibitors, PI3K and mTOR inhibitors). We developed a machine learning approach using a combination of neural networks and Bayesian network propagation analysis to identify pathway activation and mechanisms of resistance associated with each of the drugs. Our approach identified 16 combinations of drugs that had different mechanisms and downstream targets. Surprisingly, we found that histone deacetylase inhibitors (HDACi, e.g. panobinostat) were predicted to be strongly synergistic in combination with JAK inhibitors (e.g. ruxolitinib). These findings were unexpected as ruxolitinib had very weak single agent effects and the JAK-STAT pathway is not thought to be specifically associated with response to HDACi. We verified the predictions of the machine learning algorithm by performing in vitro combination assays in six different cell lines. In each case, we found that the combination was highly synergistic using the Chou-Talalay method. We further verified the feasibility and efficacy of combining panobinostat (HDACi) and the JAK inhibitor ruxolitinib in vivo using xenograft models. Both single agents had relatively modest effects on tumor burden, but we found significant synergy with the combination (p<0.01), with vastly decreased tumor burdens. In vivo modeling also allowed for testing for hematological toxicity. Hemoglobin levels and ANC remained constant with all therapies, though combination therapy caused a 25% decrease in platelet levels, which would be considered clinically tolerable with monitoring. We further performed mechanistic experiments that demonstrate that JAK-STAT pathway activation through genetic mutations in STAT3 directly contribute to HDACi resistance and reverse sensitivity to HDACi. Conclusions These results provide a powerful proof of principle for the application of large scale perturbation approaches combined with machine learning to identify novel drug combinations and mechanisms of resistance. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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Gene Expression Studies Reveal a Potential Mechanism of Glivec Resistance in Patients with Ph+ ALL Treated.

Blood ◽

10.1182/blood.v106.11.4374.4374 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4374-4374

Author(s):

To Ha Loi ◽

David D.F. Ma

Keyword(s):

Gene Expression ◽

Cell Differentiation ◽

B Cell ◽

Cell Lines ◽

Unexpected Finding ◽

B Cell Differentiation ◽

Down Regulation ◽

Blast Cells

Abstract In Ph+ ALL, patients respond to Glivec but nearly always relapse due to acquired resistance. The biological processes associated with Glivec resistance in Ph+ ALL are yet to be fully elucidated. The aim of this study is to use microarray and Q-PCR technology to dissect changes in signaling pathways of blast cells in Ph+ ALL patients treated with Glivec. Peripheral blood (PB) samples were taken before treatment and on consecutive days after administration of Glivec from two Ph+, c-ALLA+ and CD19+ ALL patients. Blast cells were isolated and their gene expression assayed using 19K cDNA microarrays. Over 400 differentially expressed genes were identified with at least a 1.5-fold up- or down-regulation in treated cells compared to cells collected pre-treatment. Based on these gene expression results, three main gene ontology groups were further evaluated: Apoptosis, Proliferation and B cell differentiation. The up-regulation of Bim and Bcl-6, and down regulation of Cyclin D2, confirms the induction of apoptosis via the FOXO3a pathway in cells treated with Glivec in vivo. Interestingly, both the proliferation genes, Tcl1-A and PKCe, and B cell differentiation associated genes, including CD79a, ETS1 and a cohort of IGH and Igl and k genes, were up-regulated during therapy. These gene expression changes observed in vivo were confirmed by Q-PCR in the Ph+ cell lines K562 (derived from CML blast crisis) and SUP-B15 (derived from ALL) treated with Glivec. The unexpected finding of increased expression of pro-proliferative genes and B cell differentiation genes by microarray revealed potential links with early B cell development and B cell receptor (BCR) signaling. Evidence for apoptosis and proliferation of Ph+ cell lines treated with Glivec were then examined by FACs. After 5 days of treatment with Glivec, 90% of K562 and 50% SUP-B15 cells underwent apoptosis. Furthermore, cell cycle analysis revealed the existence of a population of cells in G2 phase even after 6 days of Glivec treatment in SUP-B15 but not K562 cells, thus providing evidence of a population of cells undergoing proliferation during Glivec treatment in vitro. In summary, our in vivo observations supported by in vitro experiments suggest that Glivec induces the majority of Ph+ ALL blasts to undergo apoptosis. However, as treatment is prolonged, a population of ALL cells escapes death and undergoes proliferation and differentiation. We hypothesise that Glivec induced differentiation and proliferation of Ph+ cells may result in the clonal enrichment of cells resistant to Glivec.

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A Comparison of Gene Expression Signatures from Breast Tumors and Breast Tissue Derived Cell Lines

Disease Markers ◽

10.1155/2001/850531 ◽

2001 ◽

Vol 17 (2) ◽

pp. 99-109 ◽

Cited By ~ 83

Author(s):

Douglas T. Ross ◽

Charles M. Perou

Keyword(s):

Gene Expression ◽

Cell Line ◽

Cell Lines ◽

Cell Biology ◽

Expression Patterns ◽

Tumor Biology ◽

Clinical Samples ◽

Analysis Tool

Cell lines derived from human tumors have historically served as the primary experimental model system for exploration of tumor cell biology and pharmacology. Cell line studies, however, must be interpreted in the context of artifacts introduced by selection and establishment of cell linesin vitro. This complication has led to difficulty in the extrapolation of biology observed in cell lines to tumor biologyin vivo. Modern genomic analysis tool like DNA microarrays and gene expression profiling now provide a platform for the systematic characterization and classification of both cell lines and tumor samples. Studies using clinical samples have begun to identify classes of tumors that appear both biologically and clinically unique as inferred from their distinctive patterns of expressed genes. In this review, we explore the relationships between patterns of gene expression in breast tumor derived cell lines to those from clinical tumor specimens. This analysis demonstrates that cell lines and tumor samples have distinctive gene expression patterns in common and underscores the need for careful assessment of the appropriateness of any given cell line as a model for a given tumor subtype.

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Mouse Models of Human Mantle Cell Lymphoma for the Study of Disease Biology and for Pre-Clinical Assessment of Experimental Treatment Approaches.

Blood ◽

10.1182/blood.v120.21.2759.2759 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2759-2759

Author(s):

Pavel Klener ◽

Magdalena Klanova ◽

Tomas Soukup ◽

Jan Molinsky ◽

Jan Zivny ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Cell Lines ◽

Single Agent ◽

Cell Receptor ◽

Ki 67 ◽

Disease Biology ◽

B Cell Receptor Signaling

Abstract Abstract 2759 Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma associated with poor prognosis. MCL animal models for the study of disease biology and for the testing of novel agents are scarce. We established and characterized various in vivo models of metastatic blastoid human MCL by tail vein injection of five MCL cell lines (Jeko-1, HBL-2, Mino, Rec-1, Granta-519) into the NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ immunodeficient mice. Untreated animals were then observed to evaluate differences in the pattern of lymphoma growth and overall survival (OS) between different cell lines. We analyzed infiltration of selected murine organs (i.e. bone marrow [BM], spleen, liver, brain, kidneys, and enlarged lymph nodes [LN]) by immunohistochemistry (IHC) (CD20, Ki-67) at four different time-points related to OS. Extent of organ infiltration with human MCL cells was estimated using the Image-Pro Plus 5.1 software within 20 samples from different organ areas. Subsequently, we analyzed gene expression of Jeko-1 and Mino cells obtained from the xenografted animals (in vivo growing cells) compared to the cells cultured in vitro (controls).MCL cells isolated from various murine organs (the BM, liver, spleen, and LN) or in vitro cultured cells were magnetically sorted by CD45-microbeads. Gene expression analyses were carried out using Illumina BeadChips, and the data were functionally clustered with DAVID Bioinformatics tool. In addition, differences in surface expression of selected antigens were compared between in vivo vs. in vitro grown MCL cells by flow cytometry. Finally, we evaluated the anti-tumor activity of single-agent chemotherapy agents (cytarabine, fludarabine, bendamustine, and cisplatin), monoclonal antibodies (rituximab, ofatumumab, bevacizumab) or targeted agents (bortezomib, temsirolimus) in Jeko-1 and Mino bearing mice. Tumor engraftment was achieved in all the cell lines tested. The median overall survival (OS) of mice xenografted with 1–10×106 MCL cells ranged from 22 to 55 days depending on the cell line used. The principal site of engraftment and proliferation niche for all MCL cell lines was the bone marrow. MCL cells disseminated to other murine organs including the spleen, liver and brain. Development of enlarged lymph nodes (peripheral, intraabdominal) and/or extranodal MCL masses (subcutaneous tumors) were associated with Mino, while infiltration of the ovaries was inconstant finding in Jeko-1 xenografted mice. Mice xenografted with Jeko-1, HBL-2 and Granta-519 showed leukemization of peripheral blood before death. Gene expression studies of Jeko-1 and Mino in vivo growing cells revealed that the genes from the “B-cell receptor signaling” and the “oxidative-phosphorylation” pathways were the most upregulated or downregulated, respectively. In vivo growing Jeko-1 cells showed upregulation of CD31/PECAM, CD37, CD38, CD44, CD164, and downregulation of podoplanin and CXCR4. In vivo growing Mino cells had upregulation of CD23, but downregulation of CD37, CD40, CD44, CD54, CD138, CXCR4, CCR7 and podoplanin. Both Jeko-1 and Mino cells isolated from the BM (but not from the spleen, liver or LN) were significantly more sensitive to cytarabine (2–4 fold) and cisplatin (2 fold) than in vitro growing controls. Single-agent therapy of Jeko-1 and Mino bearing mice with either a chemotherapy agent, monoclonal antibody, or targeted agent resulted in significant prolongation of OS compared to untreated controls. Treatment of Jeko-1 and HBL2 bearing mice with single-agent cisplatin, single-agent cytarabine or combination of both agents revealed that the therapy with single-agent cisplatin was associated with the longest prolongation of OS. Moreover, IHC analyses of the BM, spleen and liver of the treated animals confirmed the most profound suppression of both MCL infiltration (CD20) and proliferation rate (Ki-67) in the single-agent cisplatin cohort compared to the other cohorts. In summary, the mouse models can be used for the study of MCL biology, as well as for preclinical assessment of experimental therapy of MCL including agents that cannot be properly tested in vitro (e.g. monoclonal antibodies, pro-drugs, anti-angiogenic agents, inhibitors of B-cell receptor signaling etc.). Financial Support: IGA-MZ NT13201-4/2012, GAUK 259211/110709, GAUK 446211, UNCE 204021, PRVOUK P24/LF1/3, PRVOUK 1–5101–280002 PVK, SVV-2012–254260507 Disclosures: No relevant conflicts of interest to declare.

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The Alkylating Histone Deacetylase Inhibitor Fusion Molecule Edo-S101 Displays Full Bi-Functional Properties in Preclinical Models of Hematological Malignancies

Blood ◽

10.1182/blood.v124.21.2100.2100 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2100-2100 ◽

Cited By ~ 3

Author(s):

Ana Alicia López-Iglesias ◽

Laura San-Segundo ◽

Lorena González-Méndez ◽

Susana Hernández-García ◽

Daniel Primo ◽

...

Keyword(s):

Single Dose ◽

Cell Line ◽

Cell Lines ◽

Hematological Malignancies ◽

Parp Cleavage ◽

Hdac Inhibition ◽

Histone 3 ◽

Xenograft Models

Abstract Background Alkylating histone deacetylase inhibitors (HDACi) enhance the anticancer efficacy of alkylators by increasing chromatin accessibility and also down regulating DNA repair. EDO-S101 is a first-in-class fusion molecule that combines DNA damaging effect of bendamustine with the pan-HDACi vorinostat. Objectives To study the bi-functional properties of EDO-S101 as an alkylating agent and a pan-HDACi in various in vitro and in vivo xenograft models of hematological malignancies. Methods In vitro inhibition of HDAC Class I and II enzymes by EDO-S101 and vorinostat was tested using an recombinant human enzymatic assay (BPS Bioscience, Enzo Life Science) and in vivo in rat peripheral blood mononuclear cells (PBMCs). The degree of inhibition was measured 1 hour following a single dose of 10–50 mg/kg i.v. and duration of inhibition over 24 hours after a single i.v. dose of EDO-S101 of 25 mg/kg. HDAC inhibition, alkylation and apoptotic activity were evaluated in vitro in myeloid (HL60 AML cell line) and lymphoid cell lines, including Daudi Burkitt’s lymphoma (BL) and a panel of 6 MM cell lines (MM1S, MM1R, RPMI-8226, RPMI-LR5, U266, U266-LR7). In vivo intra-tumor effects were analyzed after short courses of treatment with EDO-S101 in MM1S human plasmacytoma (PC) and BL xenograft models. Changes in pathway activation, protein expression and activities influencing the cell cycle were measured by Western blot and immunohistochemistry. Anti-tumor activity in vitro was measured by MTT and in vivo using a caliper to assess tumor size at regular intervals. Results In vitro, EDO-S101’s pan-HDACi activity, at nanomolar concentrations in Class I and II recombinant enzymes, was similar to vorinostat. In vivo, in intact rat PBMCs, HDAC inhibition was maximal at 1 hour after a single dose of 10 mg/kg i.v.–the dose where antitumor activity starts. HDAC inhibition did not increase with doses up to 50 mg/kg, recovery began within 3 hours and was nearly complete at 16 hours. In the AML HL60 cell line in vitro, hyperacetylation of lysine residues K9, K14, K23 and K56 on histone 3 was found after exposure to 2–4 µM of EDO-S101. Histone 3 and 4 hyperacetylation was also demonstrated in MM cell lines at 1–5 µM concentrations. In xenograft models of human plasmacytoma and BL, EDO-S101 induced histone 3 hyperacetylation, indicating an HDACi effect in vivo. Alkylating activity was demonstrated in vitro in HL60 and MM cell lines by DNA cross-linking and double strand break formation in the comet assay by immunofluorescence. In vivo, in xenograft models of human plasmacytoma (60 mg/kg d 1, 8, 15) and BL (40 and 80mg/kg d1) exposure to EDO-S101 caused a strong DNA-repair response shown by activation of pH2AX and p53 (PC and BL) followed by an increase of DNA damage check point proteins pCHK1 (PC) and even more prominent pCHK2 (PC and BL). The kinetics of this effect, studied in vivo in BL tumors, showed that the pH2AX response fell at Day 8 after dosing while the p53 response lasted, particularly in the group treated with 80mg/kg. In Daudi-bearing mice tumors, p-ATR was completely suppressed at Day 8 after treatment, which was not clear in the PC tumors. EDO-S101 triggered apoptosis in vitro and in vivo, resulting in strong antitumor activity in HL60, Daudi and the panel of six MM cell lines. Initial in vitro experiments in HL60 cells showed an activation of the intrinsic pathway of apoptosis with cleavage of caspases 3, 9 and PARP and a marked reduction of anti-apoptotic proteins XIAP and Mcl-1. In the MM cell line, MM1S activation of the intrinsic and extrinsic pathways of apoptosis (C 8, 9, 3, 7 and PARP cleavage) was seen with a loss of mitochondrial membrane potential by DiOC6. Tumors of human plasmacytoma and BL in vivo were rapidly shrinking or completely eradicated after i.v. administration of EDO-S101. A decrease in proliferation (Ki67) and slight PARP cleavage was found in the tumor tissue (PC), and evidence of activation of apoptosis by cleavage of caspases 7 and 9 at Day 4 and caspase 8 and PARP at Day 8 after treatment in BL tumors. The level of caspase 3, different to MM, remained unchanged. Importantly, EDO-S101 induced a rapid and dose-dependent strong decrease of XIAP and Mcl-1 which lasted until Day 8. Conclusions This study demonstrates the bi-functional mechanism of ED0-S101 in both myeloid and lymphoid hematological malignancies. The data support the clinical investigation of EDO-S101 in treating hematological malignancies. Disclosures Ocio: Mundipharma: Honoraria, Research Funding. Mehrling:Mundipharma: Employment.

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