Induction of Resistance to Proteasome Inhibition Preferentially Switches Survival Dependence from Bcl-2 to XIAP in Preclinical Models of Waldenstrom Macroglobulinemia: Pre-Clinical Rationale for Early Clinical Sequencing of ABT199

Abstract Background: The proteasome is an established and druggable target for the treatment of plasma cell-related malignancies including Waldenstrom macroglobulinemia (WM). WM cells as a consequence of high immunoglobulin production and increased B-cell receptor (BCR) mediated proliferation upregulate proteasome activity. Additionally, microenvironmental influence mediated through BCR signaling directly influences Bcl-2 and its BH3 family members, supporting tumor cell survival. Indeed, WM patients derive significant clinical benefit from proteasome-inhibitor (PI) based therapy with agents such as bortezomib and carfilzomib. However, resistance to PI develops over time and for these patients the optimal choice and sequence of therapy has yet to be determined. Using our WM models of PI-resistance we interrogated molecular events within the BCR and Bcl-2 pathways to determine therapeutic potential of targeting these crucial pathway in PI-resistance. Materials & Methods: WM cell lines (BCWM.1 and MWCL-1) and carfilzomib-resistant (CR) subclones (BCWM.1/CR and MWCL-1/CR) were used in experiments. Gene-expression and long-noncoding (LNC) RNA analysis was performed (Arraystar) and validated by real-time PCR. Bortezomib, carfilzomib, ABT199 and ibrutinib were purchased from Sellekchem. Results: To determine the functional impact of BCR and Bcl-2 signaling in PI-resistance, as well as therapeutic sensitivity of PI-resistant cells to their inhibitors (ibrutinib, ABT-199, respectively), we established and characterized WM cell lines resistant to carfilzomib. BCWM.1/CR cells showed approximately 20-fold resistance to carfilzomib (IC50 = 92.75nM) and MWCL-1/CR cells approximately 10-fold resistance (Fig. 1A). Both CR clones also displayed some cross-resistance to bortezomib. Gene expression and LNC-RNA profiling demonstrated several changes between carfilzomib-resistant vs. sensitive WM cells. Analysis of proteasome-related mRNA revealed downregulation of PSMB5, PSMB1, PSMB2 and PSMB8. Similarly, profiling of BCR-associated genes demonstrated decreased expression of several components, including BTK and SPI1. This observation functionally manifested as reduced sensitivity to the BTK-inhibitor ibrutinib, wherein CR cells displayed 1.5 - 2 fold growth resistance to ibrutinib on 72hr MTS assay. Next we examined the expression of Bcl-2 family members in CR cells. Intriguingly, we observed that Bcl-2 and Mcl-1 were significantly downregulated but XIAP (inhibitor of apoptosis) was significantly increased in CR cells vs. wildtype WM cells- both at transcriptional and translational levels. This suggested that upon acquisition of CR, a transcriptional shift towards XIAP occurs to accommodate sustained therapeutic stress from carfilzomib and maintain steady antiapoptotic composure. To test if the PI-resistant cells have moved away from their survival dependence on Bcl-2, we treated CR cells to increasing concentrations of the Bcl-2-specific inhibitor, ABT199, and as anticipated, CR cells displayed reduced apoptosis in presence of ABT199 compared to wildtype WM cells (32% annexin-V staining vs. 50%, respectively) (Fig. 1B). Conclusions: Our study sheds insight into the differential drivers of PI-resistance particularly towards carfilzomib, in WM cells. We demonstrate that acquisition of CR is associated with downregulation of Bcl-2 and Mcl-1, which is countered by upregulation of XIAP- an event that renders CR cells resistant to ABT199 (as it targets only Bcl-2). Likewise, downregulation of BCR-related components in CR cells was associated with reduced sensitivity towards ibrutinib. These observations suggest that acquisition of resistance to PI such as carfilzomib can impact future treatment with agents such as ibrutinib or ABT199. Our preclinical models provide rationale or early sequencing of ibrutinib or ABT199 in therapeutic planning of WM patients prior to induction of PI resistance. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Mir-155 Expression Raises the Apoptotic Threshold in Waldenström Macroglobulinemia By Inhibition of FOXO3a and Bim

Blood ◽

10.1182/blood.v124.21.1671.1671 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1671-1671 ◽

Cited By ~ 1

Author(s):

Brian T Gaudette ◽

Lawrence H. Boise

Keyword(s):

Cell Line ◽

Cell Lines ◽

Conflicts Of Interest ◽

Therapeutic Index ◽

Fold Increase ◽

Malignant Cell ◽

Waldenström Macroglobulinemia ◽

Waldenstrom Macroglobulinemia ◽

Apoptotic Proteins ◽

Induced Apoptosis

Abstract Waldenström Macroglobulinemia (WM) is a proliferative disorder of lymphoplasmacytic cells in the lymph nodes and bone marrow. Phenotypically, WM cells are CD19+, CD20+, CD22+, CD38+, CD138+/- and are lymphoid or plasmacytic in morphology. The disease is characterized by abundant secretion of monoclonal, IgM which causes much of morbidity associated with WM. The disease carries a high prevalence of activating somatic mutations in MyD88 (91%) and CXCR4 (28%), which have been shown to contribute to poor prognosis. These mutations involve signaling cascades that activate pathways known to enhance survival signaling including Bcl-xL. Generally, upregulation of pro-apoptotic Bcl-2 family proteins is observed as cancer cells break differentiation and proliferation checkpoints. To counter this, it becomes necessary for the cell to increase expression of anti-apoptotic Bcl-2 proteins making it dependent on a particular protein or set of proteins for survival. However, we have previously shown data that Bcl-2 family expression in WM is characterized by low expression of both pro- and anti-apoptotic proteins. To investigate a mechanism for this regulation, we examined the Bcl-2 family expression in three WM cell lines and observed that in two lines, BCWM.1 and MWCL-1, the pro-apoptotic BH3-only protein Bim was expressed at very low levels or absent, respectively, which corresponded with low sensitivity to inducers of Bim-dependent intrinsic apoptosis including ABT-737 and dexamethasone. These cell lines were sensitive to bortezomib which can induce apoptosis independent of Bim via a tBid-dependent mechanism. In the third WM cell line, RPCI-WM1, Bim was expressed at moderate levels but the pro-apoptotic proteins Bak and Bax were underexpressed and absent, respectively, which rendered the cell line completely apoptosis-deficient. Having ruled out genomic copy number variation at the loci corresponding to these genes and finding no evidence of epigenetic silencing by methylation, we examined the expression of microRNAs targeting these genes. We first examined the predicted targets of seven commonly dysregulated microRNAs in WM. Of these only one, miR-494, was found to have a moderately conserved target site in the 3’ UTR of Bim. However, the expression pattern of miR-494 did not correlate with the pattern of Bim expression in the WM cell lines. None of these microRNAs were predicted to target Bax or Bak. Therefore, we examined the expression of the remaining commonly dysregulated microRNAs and found that miR-155 was expressed at much higher levels in BCWM.1 and MWCL-1 than in RPCI-WM1 or the multiple myeloma (MM) cell line MM1.s. miR-155 is known to both directly and indirectly regulate FOXO3a, a transcription factor important in the induction of Bim. Confirming this, we observed low protein expression of FOXO3a in both BCWM.1 and MWCL-1 cells. To test this mechanism we stably expressed an anti-miR that targets miR-155 or a control anti-miR in all three WM cell lines and observed an increase in mRNA for FOXO3a and Bim as well as an increase in Bim protein in BWCM.1 and MWCL-1 cells expressing anti-miR-155, while no effect on Bim was observed in the RPCI-WM1 line that does not express miR-155 at high levels. This corresponded with a two-fold increase in ABT-737-induced apoptosis in both BWCM.1 and MWCL-1 in the absence of any additional death signal. As expected, miR-155 antagonism did not significantly increase bortezomib-induced apoptosis. These data indicate that miR-155 expression raises the apoptotic threshold in WM by limiting FOXO3a-mediated Bim expression. Cancer therapy relies on the ability to kill malignant cells at a lower dose than would kill healthy cells. This therapeutic index relies heavily on what is termed mitochondrial priming which is a measure of the expression of pro-apoptotic proteins in a cell. The malignant cell remains alive due to sequestration of these proteins by anti-apoptotic proteins, yet requires less death signaling to cause release of sufficient quantities of pro-apoptotic proteins to activate apoptosis. The data presented here indicate that increased expression of miR-155 raises the apoptotic threshold of WM cells by inhibiting Bim expression and thereby compromises the therapeutic index of many agents. Therefore, the sensitivity to a variety of apoptosis-inducing therapies would be increased by targeting miR-155 in combination as part of the treatment modality. Disclosures No relevant conflicts of interest to declare.

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Protein citrullination as a source of cancer neoantigens

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-002549 ◽

2021 ◽

Vol 9 (6) ◽

pp. e002549

Author(s):

Hiroyuki Katayama ◽

Makoto Kobayashi ◽

Ehsan Irajizad ◽

Alejandro Sevillarno ◽

Nikul Patel ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Immune Response ◽

Protein Expression ◽

Cell Lines ◽

Cancer Cell ◽

Family Members ◽

Immunohistochemical Analysis ◽

Cancer Cell Lines ◽

Mhc Ii

BackgroundCitrulline post-translational modification of proteins is mediated by protein arginine deiminase (PADI) family members and has been associated with autoimmune diseases. The role of PADI-citrullinome in immune response in cancer has not been evaluated. We hypothesized that PADI-mediated citrullinome is a source of neoantigens in cancer that induces immune response.MethodsProtein expression of PADI family members was evaluated in 196 cancer cell lines by means of indepth proteomic profiling. Gene expression was assessed using messenger RNA data sets from The Cancer Genome Atlas. Immunohistochemical analysis of PADI2 and peptidyl-citrulline was performed using breast cancer tissue sections. Citrullinated 12–34-mer peptides in the putative Major Histocompatibility Complex-II (MHC-II) binding range were profiled in breast cancer cell lines to investigate the relationship between protein citrullination and antigen presentation. We further evaluated immunoglobulin-bound citrullinome by mass spectrometry using 156 patients with breast cancer and 113 cancer-free controls.ResultsProteomic and gene expression analyses revealed PADI2 to be highly expressed in several cancer types including breast cancer. Immunohistochemical analysis of 422 breast tumor tissues revealed increased expression of PADI2 in ER− tumors (p<0.0001); PADI2 protein expression was positively correlated (p<0.0001) with peptidyl-citrulline staining. PADI2 expression exhibited strong positive correlations with a B cell immune signature and with MHC-II-bound citrullinated peptides. Increased circulating citrullinated antigen–antibody complexes occurred among newly diagnosed breast cancer cases relative to controls (p=0.0012).ConclusionsAn immune response associated with citrullinome is a rich source of neoantigens in breast cancer with a potential for diagnostic and therapeutic applications.

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Compound-specific adaptation of hepatoma cell lines to toxic iron

Metallomics ◽

10.1039/c9mt00202b ◽

2019 ◽

Vol 11 (11) ◽

pp. 1836-1846

Author(s):

Sarah Guttmann ◽

Elisabeth Therese Dewald ◽

Cathrin Wohlfarth ◽

Jennifer-Christin Müller ◽

Uwe Karst ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Hepatoma Cell ◽

Hepatic Gene Expression ◽

Specific Adaptation ◽

Hepatoma Cell Lines ◽

Resistant Cells

Toxic iron exposure induces broad modulation of hepatic gene expression and establishment of resistant cells.

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Activation of the Wnt/β-Catenin Pathway Mediates Lenalidomide Resistance in Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.113.113 ◽

2009 ◽

Vol 114 (22) ◽

pp. 113-113 ◽

Cited By ~ 1

Author(s):

Chad C. Bjorklund ◽

Deborah J. Kuhn ◽

Jairo A. Matthews ◽

Michael Wang ◽

Veerabhadran Baladandayuthapani ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Cell Lines ◽

Expression Profiling ◽

Fold Increase ◽

Resistant Cell ◽

Refractory Disease ◽

Wild Type ◽

Myeloma Cells ◽

Resistant Cells

Abstract Abstract 113 Background: Novel drugs such as the immunomodulatory agent lenalidomide have revolutionized the treatment of multiple myeloma, as evidenced by an increasing overall survival for patients with both newly-diagnosed, and relapsed and/or refractory disease. Despite these improvements, myeloma remains incurable, and is still characterized by a trend for increasing chemoresistance at relapse, with a decreasing duration of benefit from each successive line of therapy. By understanding the mechanisms responsible for the emergence of drug resistance, which have so far not been well characterized in the case of lenalidomide, it may be possible to rationally design novel regimens that could either overcome this resistance, or possibly prevent its emergence altogether. Methods: To improve our understanding of the mechanisms responsible for lenalidomide resistance, we developed cell line models of interleukin (IL)-6-dependent (ANBL-6 and KAS-6/1) and –independent (U266 and MM1.S) lenalidomide-resistant multiple myeloma cells. Starting at a concentration that was 1/10 of the IC50 for lenalidomide's anti-proliferative effects in drug-naïve cells, increasing drug concentrations were used until all the cell lines could proliferate and maintain cell membrane integrity in the presence of 10 μM lenalidomide. These cell lines were then used as an in vitro model of lenalidomide-specific drug resistance, and subjected to further characterization, including with gene expression profiling. Results: Resistance to lenalidomide was evidenced by a dramatic, 100-1000-fold increase in the IC50 values of these myeloma cells. In the case of ANBL-6 cells, for example, drug-naïve cells showed an IC50 of 0.14 μM using tetrazolium dye-based viability assays, but this increased to >100 μM in the drug-resistant cells, as was the case in U266 and MM1.S cells. This resistance was a stable phenotype, since removal of lenalidomide for seven to ninety days from cell culture conditions did not re-sensitize them when 10 μM lenalidomide was reintroduced. Gene expression profiling followed by pathway analysis to examine changes at the transcript level between wild-type parental and lenalidomide-resistant cell lines identified the Wnt/β-catenin pathway as the most altered across all cell lines. Increased expression was seen in several members of the low-density-lipoprotein receptor related protein family, including LRP1 and 5; members of the wingless-type MMTV integrations site family, including WNT3 and 4; β-catenin; and downstream Wnt/β-catenin targets such as CD44. Similar changes were detected in primary samples from a patient who developed clinically lenalidomide-refractory disease. Reporter assays revealed an up to 5-fold increase in LEF/TCF-dependent transcription both in drug-naïve cells acutely exposed to lenalidomide, and in their chronically exposed, lenalidomide-resistant clones. Western blotting and flow cytometry confirmed that these lenalidomide-resistant cells had increased expression by 2-20 fold of β-catenin and CD44, as well as other LEF/TCF targets, including Cyclin D1 and c-Myc. Comparable changes occurred after lenalidomide exposure in myeloma cells grown in the context of bone marrow stroma. Notably, lenalidomide-resistant cells showed decreased expression of casein kinase 1 and increased phosphorylation of glycogen synthase kinase 3 at Ser21/9, both of which would reduce the phosphorylation of β-catenin needed for its later proteasome-mediated degradation. Stimulation of the Wnt/β-catenin pathway with recombinant human Wnt3a resulted in resistance to lenalidomide in wild-type, drug-naïve cells, as evidenced by a 10-fold increase in the IC50. Conversely, exposure of lenalidomide-resistant cell lines to quercetin, a known antagonist of the β-catenin/TCF interaction, induced a partial re-sensitization to lenalidomide. Conclusions: These data support the hypothesis that activation of the Wnt/β-catenin pathway represents a mechanism of both acute and chronic resistance to the anti-proliferative effects of lenalidomide in multiple myeloma. Moreover, they support the development of strategies aimed at suppressing Wnt/β-catenin activity to resensitize multiple myeloma to the effects of this immunomodulatory agent in vivo. Disclosures: No relevant conflicts of interest to declare.

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Carfilzomib Exerts Anti-Neoplastic Activity in Waldenstrom Macroglobulinemia.

Blood ◽

10.1182/blood.v114.22.4916.4916 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4916-4916

Author(s):

Antonio Sacco ◽

Aldo M. Roccaro ◽

Monette Aujay ◽

Hai Ngo ◽

Feda Azab ◽

...

Keyword(s):

Cell Lines ◽

Low Grade ◽

Dependent Manner ◽

Caspase 9 ◽

Employment Equity ◽

Waldenström Macroglobulinemia ◽

Waldenstrom Macroglobulinemia ◽

Synergistic Cytotoxicity ◽

Equity Ownership ◽

Dose Dependent

Abstract Abstract 4916 Introduction Proteasome inhibition represents a valid therapeutical approach in several tumors and its use has been validated in Waldenstrom's macroglobulinemia (WM), where single-agent Bortezomib has been successfully tested in phase 2 clinical trials. Nevertheless, a significant fraction of patients relapse, or develop significant toxicity due to high toxicity in non-transformed cells. Therefore preclinical evaluation of new proteasome inhibitor with a more selective inhibition of neoplastic cells is needed in order to increase efficacy and improve patient outcome. We tested Carfilzomib, a tetrapeptide epoxyketone selective inhibitor of the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome in WM. Methods WM and IgM secreting low-grade lymphoma cell lines (BCWM.1, MEC1, RL) were used. Expression of imunoproteasome and constitutive proteasome subunits (beta1, beta2, beta5; LMP2, MECL1, LMP7) were detected primary WM cells and cell lines by an ELISA-based assay. Cytotoxicity and DNA synthesis were measured by thymidine uptake and MTT, respectively. Cell signaling and apoptotic pathways were determined by Western Blot. Determination of the additive or synergistic effect of drugs combination was calculated using the CalcuSyn software based on the Chou-Talalay method. Results Primary CD19 bone-marrow derived WM cells express higher level of the immunopreoteasome as compared to the constitutive proteasome. Carfilzomib inhibited the chymotrypsin-like activity of both the immunoproteasome (LMP7) and the constitutive proteasome (beta5) and in WM cells, in a dose-dependent manner; leading to inhibition of proliferation (IC50: 5nM; 48h) and induction of cytotoxicity (IC50: 7.5nM; 48h) in WM cells. Carfilzomib mediated apoptosis in WM by increasing PARP-, caspase-9- and -3-cleavage; as well as by inducing activation of c-jun-N-terminal kinase and ER-stress in a dose-dependent manner. Moreover, combination of Carfilzomib and bortezomib induced synergistic cytotoxicity in WM cells, as shown by enhanced PARP-, caspase-9- and -3-cleavage; and synergy in inhibiting the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome. Conclusion Taken together, these findings provide the pre-clinical rational for testing Carfilzomib in Waldenstrom Macroglobulinemia. Disclosures Aujay: Proteolix: Employment, Equity Ownership. Demo:Proteolix: Employment, Equity Ownership. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

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PIM as a Rational Target for B-Cell Lymphomas.

Blood ◽

10.1182/blood.v114.22.3946.3946 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3946-3946

Author(s):

Cristina Gomez-Abad ◽

Helena Pisonero ◽

Juan F Leal ◽

Giovanna Roncador ◽

Jose A. Martinez-Climent ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Specific Inhibitor ◽

B Cell Lymphoma ◽

Large B Cell Lymphoma ◽

Micromolar Range ◽

Large B Cell ◽

Stat Pathway

Abstract Abstract 3946 Poster Board III-882 INTRODUCTION The Pim kinases are a family of serine/threonine kinases composed by three members: Pim1, Pim2 and Pim3, involved in the phosphorylation and regulation of several proteins that are essential for cell cycle progression, metabolism or apoptosis (BAD, p21, p27KIP, AKT, Mdm2 and cMyc, among them). Overexpression, translocation or amplification of Pim family have been described in many human cancers, including B-cell Non Hodgkin's Lymphoma, Multiple Myeloma, Prostate cancer and Pancreatic cancer. In addition, 50% of patients diagnosed with diffuse large B-cell Lymphoma (DLBCL) present somatic mutations in Pim1. Despite of its important role in cancer progression, very few chemical inhibitors have been described in the literature, being effective all of them in the high micromolar range. PURPOSE Validating PIM as a rational therapeutic target in B-cell lymphoma, developing tools for patient stratification and pharmacodynamic studies on PIM inhibition. MATERIAL AND METHODS Gene expression profiling and Copy Number data were obtained from a series of 94 B-cell Non-Hodgkin Lymphoma patients (DLBCL, FL, MALT, MCL and NMZL). The effect of Pim inhibition was checked on cell lines by using a novel specific inhibitor for the Pim family (ETP-39010). Newly produced antibodies and RT-PCR primers and protocols were standarized. RESULTS Gene expression data revealed high Pim isoforms expression in a subset of patients with Mantle cell lymphoma (MCL), and Diffuse Large B-cell lymphoma (DLBLC)-ABC type. CGH analysis focused on chromosomal regions containing Pim family and its main regulatory upstream pathway (JAK/STAT) was performed. Heterozygous gains of Pim1 (6p21.2) and Pim3 (22q13.33) were identified in 13.6% of DLBCL patients and in 4.2% of MCL. Alterations in JAK/STAT pathway were also detected in 59.1% of DLBCL patients, and 37.5% of MCL patients presented any alteration in JAK/STAT pathway, being frequent losses of JAK2 chromosomal region. Analysis of additional pathways involved in the up-stream regulation of Pim family disclosed heterozygous gains of PIK3C3 in 40.9% of DLBCL patients, and gains of PIK3CA in 45.9% of MCL patients. Lymphoma cell lines (15) derived from both MCL (9) and ABC-DLBLC (6) subtype, have been analyzed by qRT-PCR and Western-blot, showing variable expression levels of Pim1, Pim2 and Pim3. IC50 obtained for the ETP-39010 compound is in the low micromolar range for the MCL (0.7-8.7 micromolar) and DLBCL-ABC (0.8-10.3 micromolar) cell lines. Since Pim kinase family phosphorilate multiple sites of Bad and AKT, we have checked the inhibition of its phosphorilation as molecular biomarkers for the ETP-39010 effect. Our data show an inhibition of at least 20% of pBad (S112) and almost a complete inhibition of pAKT (S473) 4h after treatment. In addition, cell cycle arrest at G1 and induction of apoptosis were observed 24h after the treatment. CONCLUSION Pim family genes are a rational therapeutic target in MCL and DLBCL-ABC lymphoma subtypes. Stratification and pharmacodynamic markers have been developed for PIM inhibition using a novel specific inhibitor compound -ETP-39010-. Disclosures: No relevant conflicts of interest to declare.

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HOXA9 Is a Novel Therapeutic Target in Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.832.832 ◽

2009 ◽

Vol 114 (22) ◽

pp. 832-832 ◽

Cited By ~ 1

Author(s):

Michael A Chapman ◽

Jean-Philippe Brunet ◽

Jonathan J Keats ◽

Angela Baker ◽

Mazhar Adli ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Histone Modification ◽

Cell Lines ◽

Therapeutic Target ◽

Copy Number ◽

Conflicts Of Interest ◽

Specific Expression ◽

Aberrant Expression ◽

High Level

Abstract Abstract 832 We hypothesized that new therapeutic targets for multiple myeloma (MM) could be discovered through the integrative computational analysis of genomic data. Accordingly, we generated gene expression profiling and copy number data on 250 clinically-annotated MM patient samples. Utilizing an outlier statistical approach, we identified HOXA9 as the top candidate gene for further investigation. HOXA9 expression was particularly high in patients lacking canonical MM chromosomal translocations, and allele-specific expression analysis suggested that this overexpression was mono-allelic. Indeed, focal copy number amplifications at the HOXA locus were observed in some patients. Outlier HOXA9 expression was further validated in both a collection of 52 MM cell lines and 414 primary patient samples previously described. To further verify the aberrant expression of HOXA9 in MM, we performed quantitative RT-PCR, which confirmed expression in all MM patients and cell lines tested, with high-level expression in a subset. To further investigate the mechanism of aberrant HOXA9 expression, we interrogated the pattern of histone modification at the HOXA locus because HOXA gene expression is particularly regulated by such chromatin marks. Accordingly, immunoprecipitation studies showed an aberrantly low level of histone 3 lysine 27 trimethylation marks (H3K27me3) at the HOXA9 locus. H3K27me3 modification is normally associated with silencing of HOXA9 in normal B-cell development. As such, it appears likely that the aberrant expression of HOXA9 in MM is due at least in part to defects in histone modification at this locus. To determine the functional consequences of HOXA9 expression in MM, we performed RNAi-mediated knock-down experiments in MM cell lines. Seven independent HOXA9 shRNAs that diminished HOXA9 expression resulted in growth inhibition of 12/14 MM cell lines tested. Taken together, these experiments indicate that HOXA9 is essential for survival of MM cells, and that the mechanism of HOXA9 expression relates to aberrant histone modification at the HOXA9 locus. The data thus suggest that HOXA9 is an attractive new therapeutic target for MM. Disclosures: No relevant conflicts of interest to declare.

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RNAi Screen of the Druggable Genome Identifies Modulators of Proteasome Inhibitor Sensitivity in Myeloma Including CDK5.

Blood ◽

10.1182/blood.v114.22.602.602 ◽

2009 ◽

Vol 114 (22) ◽

pp. 602-602

Author(s):

Yuan Xiao Zhu ◽

Rodger E. Tiedemann ◽

Chang-Xin Shi ◽

Jessica Schmidt ◽

Laura Bruins ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Conflicts Of Interest ◽

Shrna Expression ◽

Primary Screening ◽

A Genome ◽

Proteasome Subunits ◽

Druggable Genome ◽

Sensitization Effect

Abstract Abstract 602 The molecular target(s) which co-operate with proteasome inhibition in inducing drug sensitivity or resistance in Multiple Myeloma (MM) remain unknown. We therefore conducted a genome scale small interfering RNA (siRNA) lethality study in KMS11 MM cells in the presence or absence of bortezomib without regard to pre-conceived mechanistic notions. Primary screening was performed in a single-siRNA-per-well format with the human druggable genome siRNA set V4 comprising 13,984 siRNA targeting 6,992 genes and comprising two RNAi per gene. siRNA were transfected at low concentration (13nM) to minimize off-target effects using conditions that resulted in transfection of >95% cells and <5% background cytotoxicity. Bortezomib was added (at the IC10, 25, 70 and 90) 24 hours post transfection. After 96 hours (72 hours after bortezomib), viability was measured by ATP-dependent luminescence. In primary screening 320 candidate bortezomib sensitizing genes were identified and rescreened with four siRNA oligos each gene. 57 of the top sensitizer hits were selected for which at least two distinct siRNA decreased the EC50 by 2 standard deviations from cells treated with control siRNAs. By cross referencing gene expression profile (GEP) data of KMS11, we furthered pared the list to 37 plausibly expressed targets (0.5% of those genes originally screened) as bortezomib sensitizers. After silencing, 50% of these 37 genes also sensitized the lung cancer cell line A549 to bortezomib. After further parsing of genes which also modulated the sensitivity of MM to Melphalan (non specific chemosensitizers), 34 genes remained: The strongest sensitizers to bortezomib were the proteasome subunits PSMA5, PSMB2, PSMB3, PSMB7 but included less obvious targets such as BAZ1B, CDK5, CDC42SE2, MDM4, NME7, TFE3, TNFAIP3, TNK1, TOP1, VAMP2 and YY1 were also identified. Of these, the most potent synergetic effects were observed with siRNAs against the proteasome subunits and against cyclin dependent kinase 5 (CDK5), which caused the greatest shift in EC50. CDK5 is of particular interest as a therapeutic target as it is expressed at high levels in MM and neural tissues but has low expression in other organs. Using viral shRNA expression, silencing CDK5 consistently increased the sensitivity of genetically variable MM cell lines (n=5) to all of the proteasome inhibitors tested: bortezomib, carfilzomib and PR047, and the effect could be at least partially rescued by overexpression of an RNAi resistant CDK5. To explore therapeutic relevance the small molecule CDK5 inhibitor, Roscovitin, was shown to be synergistic or additive with bortezomib in both MM cell lines and primary patient samples. Gene expression profiling was then performed to seek an explanation for the CDK5 sensitization effect and regulation of a proteasome subunit PSMB5 by CDK5 was identified as a probable route to sensitization. In summary inhibition of the existing proteasome either directly by suppression of proteasome subunits, or indirectly by suppression of modulators such as CDK5 appears to confer the greatest sensitization effect suggesting that combinations of bortezomib with other unique proteasome inhibitor drugs or combinations with inhibitors of CDK5 is a logical avenue for clinical exploration. Disclosures: No relevant conflicts of interest to declare.

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CBX8, a Polycomb-Group Protein, Is Essential for MLL-AF9-Induced Leukemogenesis

Blood ◽

10.1182/blood.v116.21.4174.4174 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4174-4174

Author(s):

Jiaying Tan ◽

Jay L. Hess

Keyword(s):

Gene Expression ◽

Cell Growth ◽

Cell Lines ◽

Transcriptional Activation ◽

Fusion Proteins ◽

Conflicts Of Interest ◽

Leukemia Cell ◽

Polycomb Group ◽

Human Leukemia ◽

Interaction Sites

Abstract Abstract 4174 Trithorax and Polycomb-group (Trx-G and Pc-G) proteins are antagonistic regulators of homeobox-containing (Hox) gene expression that play a major role in regulation of hematopoiesis and leukemogenesis. Mixed lineage leukemia (MLL), a mammalian Trx-G protein, is a histone methyltransferase crucial for embryonic development and hematopoiesis that is commonly altered by translocation in acute leukemia. Recent evidence suggests that transformation by MLL fusion proteins is dependent on multiple interaction complexes, including the polymerase associated factor complex (PAFc) and the elongation activating protein complex (EAPc) or a closely related AF4 family/ENL family/P-TEFb complex (AEPc). CBX8 is a human PcG protein, functioning as a transcription repressor in the polycomb repressive complex 1 (PRC1). Previous studies have shown that CBX8 also interacts with the EAPc components AF9 and ENL; however, its role in leukemogenesis is unknown. To elucidate the significance of this interaction between these two proteins thought to have antagonistic function, we generated a large series of point mutations in AF9 and identified two amino acids that are essential for CBX8 interaction but preserve the interaction with other EAP components. Mutation of the two sites reduced the transcriptional activation of the MLL-AF9 target promoters by nearly 50% and completely inhibits the ability of MLL-AF9 to immortalize bone marrow (BM) as assessed by methylcellulose replating assays. This finding suggests that CBX8 interaction is essential for MLL-AF9-induced leukemogenesis. Several lines of evidence further support this finding. First, CBX8 knockdown by siRNAs decreased MLL-AF9-induced transcriptional activation by approximately 50%. Second, the ability of MLL-AF9 to transform primary BM was markedly reduced by retroviral shCbx8 transduction. Notably, this inhibitory effect is specific for MLL-AF9 because the BM transformation ability of E2A-HLF was unaffected by Cbx8 suppression. Third, Cbx8 suppression by shCbx8 in MLL-AF9 and MLL-ENL, but not E2A-HLF transformed AML cell lines, significantly inhibited the expression of MLL-dependent target genes, as well as cell growth and colony forming ability. Fourth, inducing CBX8 knockdown in human leukemia cell lines expressing MLL-AF9 led to a marked decrease in the localization of basic transcription machinery at the Hoxa9 locus and a corresponding reduction in Hoxa9 transcription. Importantly, the observed effects of CBX8 on MLL-rearranged leukemia cells are PRC1-independent: no effects on MLL target gene expression, cell growth, or BM transformation ability were observed by suppressing other core components of PRC1. Taken together, our results indicate that CBX8, independent of its transcription repression role in PRC1, interacts with and synergizes with MLL fusion proteins to promote leukemogenesis. Defining the interaction sites between AF9/ENL and CBX8 and the dependence of other AML subtypes and normal hematopoiesis on CBX8 will be important for the further development of agents that target this mechanism in MLL-rearranged and potentially other AML subtypes. Disclosures: No relevant conflicts of interest to declare.

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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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