A2A or Beta-2 Adrenergic Receptor Agonist Synergies Induce Distinct Patterns of Gene Expression Relevant to Multiple Myeloma Cell Survival.

Abstract Abstract 3831 Poster Board III-767 Using a high-throughput combination screening strategy, we have demonstrated that adenosine A2A receptors (A2AR) or beta-2 adrenergic receptors (bAR) agonists, in low nano-molar range, exhibit highly synergistic anti-proliferative activity when combined with Multiple Myeloma drugs, such as dexamethasone, lenalidomide, bortezomib, melphalan and doxorubicin at clinically relevant concentrations. Synergy and selectivity have been observed in multiple myeloma (MM) cell lines and ex vivo using patient tumor cells. To understand this synergistic mechanism, we employed the Affymetrix U133 plus 2.0 cDNA microarray to investigate the differentially expressed genes in a multiple myeloma MM1.S cell line treated with A2AR agonists, bAR agonists, dexamethasone, or in combinations (A2AR/ bAR agonists and dex) for six hours. Using conventional hierarchical clustering, unsupervised analyses clearly separate the A2AR/ bAR agonists or the dexamethosone groups from the combination groups, suggesting a unique mechanism underlying the combination treatments. With SAM (Significance Analysis of Microarrays), we found 314 and 309 genes that showed statistically significant up-regulation or down-regulation respectively in the combination groups compared to the untreated control, with a false discovery rate=<1%. Interestingly, only a few genes were statistically different between A2AR agonists with dex and bAR agonists with dex, indicating that they may function with a similar mechanism. To further investigate the genes that are coordinately expressed, we performed gene set enrichment analyses (GESA) with the published or curated gene sets available from Board Institute (Cambridge, MA) or GeneGo pathway analysis software (Encinitas, CA). We found that the cells treated with drug combinations were enriched in genes involved in multiple cancer relevant pathways including TGF-beta receptor signaling, WNT signaling, MAPK/Erk signaling, PKA signaling, IGF-RI signaling, the stress response pathway and the myc pathway. Bim1 is one of the genes upregulated by single agent and combination drug treatment and is important for cell killing as siRNA targeting Bim1 reduces single agent and combination effects. Down-regulation of IRF4 by combination treatments is particularly interesting. High levels of IRF-4 expression have been suggested as an independent risk factor for poor survival in multiple myeloma patients (Heintel D et. al., Leukemia 2008). Moreover, IRF4 has recently been shown to be critical for proliferation and/or survival in a number of multiple myleoma cell lines (Shaffer et al., Nature 2008). Consistent with these observations, flow cytometric analysis of annexinV/PI stained MM1.S cells transfected with siRNA against IRF4 confirms that IRF-4 is important for MM.1S survival. Furthermore, direct or indirect downstream targets of IRF4 (e.g. MYC, HK2, PDK1, and SUB1) are coordinately down-regulated in MM1.S cells when IRF4 expression is reduced by A2AR / bAR agonists with dex. These data suggest that A2A and bAR agonist combinations coordinately down-regulate both myc and IRF4, which form a positive autoregulatory loop important for MM proliferation/survival and further underscore the utility of systematic combination screening in the identification of pathway and chemical interactions relevant to disease. Disclosures: Tam: CombinatoRx, Inc.: Employment. Rickles:CombinatoRx, Inc.: Employment. Giordano:CombinatoRx, Inc.: Employment. Borisy:CombinatoRx, Inc.: Employment. Lee:CombinatoRx, Inc.: Employment.

Download Full-text

Efficacy and Safety of Daratumumab Combined With All-Trans Retinoic Acid in Relapsed/Refractory Multiple Myeloma

Blood Advances ◽

10.1182/bloodadvances.2021005220 ◽

2021 ◽

Author(s):

Kristine A. Frerichs ◽

Monique Christina Minnema ◽

Mark-David Levin ◽

Annemiek Broijl ◽

Gerard MJ Bos ◽

...

Keyword(s):

Multiple Myeloma ◽

Stable Disease ◽

Immune Cell ◽

Ex Vivo ◽

Phase 1 ◽

Single Agent ◽

Flow Cytometric Analysis ◽

Optimal Dose ◽

Efficacy And Safety ◽

Cd38 Expression

The efficacy of daratumumab is partially dependent on CD38 expression on multiple myeloma (MM) cells. We have previously shown that ATRA upregulates CD38 expression and reverts daratumumab-resistance ex vivo. We therefore evaluated the optimal dose, efficacy and safety of daratumumab combined with ATRA in daratumumab-refractory MM patients in a phase 1/2 study (NCT02751255). In part A of the study, 63 patients were treated with daratumumab monotherapy. Fifty daratumumab-refractory patients were subsequently enrolled in part B, and treated with daratumumab (re-intensified schedule) combined with ATRA until disease progression. The recommended phase 2 dose of ATRA in combination with daratumumab was defined as 45 mg/m2. At this dose, the overall response rate (ORR) was 5%, indicating that the primary endpoint (ORR≥15%) was not met. However, the majority of patients (66%) achieved at least stable disease. After a median follow-up of 43 months, the median PFS for all patients was 2.8 months. Patients who previously achieved at least a partial response or minimal response/stable disease with prior daratumumab monotherapy had a significantly longer PFS, compared to those who immediately progressed during daratumumab as single agent (median PFS 3.4 and 2.8 versus 1.3 months). The median OS was 19.1 months. The addition of ATRA did not increase the incidence of adverse events. Flow cytometric analysis revealed that ATRA temporarily increased CD38 expression on immune cell subsets. In conclusion, the addition of ATRA and re-intensification of daratumumab had limited activity in daratumumab-refractory patients, which may be explained by the transient upregulation of CD38 expression.

Download Full-text

Adenosine A2A and Beta-2 Adrenergic Receptor Agonism: Novel Selective and Synergistic Multiple Myeloma Targets Discovered through Systematic Combination Screening

Blood ◽

10.1182/blood.v112.11.384.384 ◽

2008 ◽

Vol 112 (11) ◽

pp. 384-384

Author(s):

Richard J. Rickles ◽

Laura Pierce ◽

Thomas Giordano ◽

Winnie F. Tam ◽

William Avery ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

High Throughput ◽

Adrenergic Receptor ◽

Molecular Mechanisms ◽

Single Agent ◽

Inhibition Of Proliferation ◽

Cgs 21680 ◽

Beta 2 ◽

Adenosine A2a

Abstract Using a high throughput combination screening strategy, we have discovered that agonism of either adenosine A2A receptors (A2A) or beta-2 adrenergic receptors (bAR) demonstrate significant, synergistic, anti-proliferative effects in preclinical Multiple Myeloma (MM) models. Using quantitative synergy analysis, we observe that A2A and bAR agonists have significant anti-proliferative effects in a broad panel of 10 MM cell lines when combined with each other or with standard MM agents. Individual A2A agonists CGS-21680 and HE-NECA inhibited proliferation 25–80% with EC50s ranging from 2–20 nM. Individual bAR agonists salmeterol and formoterol inhibited proliferation 35–75% with EC50s ranging from 10–30 pM. Potent, highly synergistic, inhibition of proliferation, up to 95%, was demonstrated with combinations of A2A or bAR agonists and multiple agents including dexamethasone, lenalidomide, bortezomib, melphalan, doxorubicin, HDAC inhibitors and HSP90 inhibitors at clinically relevant concentrations. These combinations exceeded Loewe additivity, and demonstrated both substantial increases in efficacy over maximal single agent levels as well as significant potency shifting with many combination indices (CIs) in the range of 0.1 to 0.3. Synergistic anti-proliferative effects were observed broadly across several MM cell lines and when using cell lines unresponsive to standard MM drugs, e.g. A2A agonists CGS-21680 and HE-NECA in combination with dexamethasone inhibited 75–85% of the proliferation of EJM, and MOLP-8 dexamethasone-insensitive cell lines as compared to 35–60% for the single agent A2A agonists. The selective A2A antagonist SCH58261 but not A1, A2B and A3 selective antagonists DPCPX, MRS1754 and MRS1523 blocked the synergy and antiproliferative activity of HE-NECA, demonstrating that the effect is mediated via the A2A receptor. siRNA directed against adenosine and adrenergic receptor isoforms, caused a concomitant reduction in the antiproliferative effects of HE-NECA and salmeterol. Synergy (CI<0.4) observed between A2A and bAR agonists suggested that while both these targets signal through Gs coupled signaling pathways, the two targets contribute to the antiproliferative effect via distinct molecular mechanisms. Anti-proliferative effects occurred through a synergistic induction of apoptosis. Combinations of either agonist with dexamethasone demonstrate 50–75% Annexin V positive MM.1S cells after 24 hours treatment whereas single agents show less than 10%. The activity, synergy and selectivity of A2A and bAR combinations were observed in xenograft models of MM. SCID CB17 mice received subcutaneous inoculation of RPMI-8226 cells and once tumors were palpable, mice were treated with vehicle, bortezomib (0.5 mg/kg IV Q3D), salmeterol (10 mg/kg s.c QD) or the combination of both agents. After 19 days of treatment, the combination showed significantly greater reduction in tumor volume than either of the single agents alone (70% vs. 34%; p<0.05, ANOVA). High throughput combination screening facilitated the discovery of two novel and related classes of drug targets with highly synergistic and selective anti-tumor activity in MM. These preclinical data provide a strong rationale for the investigation of A2A and bAR agonists in the treatment of MM.

Download Full-text

A Selective PIM Kinase Inhibitor Is Highly Active In Multiple Myeloma: Mechanism of Action and Signal Transduction Studies

Blood ◽

10.1182/blood.v116.21.4084.4084 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4084-4084 ◽

Cited By ~ 7

Author(s):

Veerendra Munugalavadla ◽

Leanne Berry ◽

Yung-Hsiang Chen ◽

Gauri Deshmukh ◽

Jake Drummond ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Combination Treatment ◽

Kinase Inhibitor ◽

Single Agent ◽

Myeloma Cell ◽

Negative Regulator ◽

Combination Drug ◽

Employment Equity ◽

Equity Ownership

Abstract Abstract 4084 Related work from our group has shown the therapeutic utility of PIM inhibition in multiple myeloma cell lines, xenografts, and primary patient samples (Ebens A. et al., ASH 2010 submitted abstr.). In this study we provide detailed mechanistic findings to show that PIM kinase inhibition co-regulates several important elements of the PI3K/AKT/mTOR pathway, resulting in significant synergy for combination drug treatments. The PIM kinases are a family of 3 ser/thr growth factor- & cytokine-induced proteins hypothesized to have redundant survival and growth functions. GNE-652 is a pan-PIM kinase inhibitor with picomolar biochemical potencies and an excellent kinase selectivity profile. Myeloma cell lines exhibit sensitivity to single agent PIM inhibition and a striking synergy in combination with the PI3K inhibitor GDC-0941. Cells respond to this combination with cell cycle arrest and marked apoptosis in vitro. We tested a panel of selective PI3K/AKT/mTOR inhibitors and found PI3K and AKT inhibitors showed the greatest extent of synergy with GNE-652, whereas mTOR inhibitors were synergistic to a lesser extent. These results suggest that PIM signaling converges on both TORC1 and AKT to generate these differential synergies. BAD is a negative regulator of both Bcl-2 and Bcl-XL, and we were able to confirm previous reports that AKT and PIM cooperate to inactivate BAD (Datt et al., 1997; Yan et al., 2003). Pim has been shown to potentially inactivate PRAS40, a negative regulator of TORC1 (Zhang et al., 2009). We demonstrate that PIM or PI3K inhibition caused a loss of phosphorylation on PRAS40 and results in a physical association of PRAS40 and TORC1 and a decrease in phosphorylated p70S6K and S6RP. These reductions were apparent in 7 of 7 cell lines assayed and enhanced by the combination of PI3K and PIM inhibition in these cell lines. Consistent with prior reports (Hammerman et al., 2005), we show that a second node of convergence between PIM and TORC1 is 4E-BP1. Both GDC-0941 and GNE-652 treatments reduced phosphorylation of 4E-BP1 in 7 of 7 myeloma cell lines. Since dephosphorylated 4E-BP1 competes with eIF4G for the mRNA cap binding factor eIF4E, we assayed immunoprecipitates of eIF4E for the presence of eIF4G and 4E-BP1 and observed increased BP1 and decreased 4G. The combination treatment significantly enhanced the loss of 4G relative to either single agent, and importantly, even at 5× the IC50 concentrations for single agents, combination drug treatment achieved greater extent of effect than single agent treatment. Thus PI3K and PIM pathways are redundant at the level of cap-dependent translational initiation mediated by eIF4E. It has been hypothesized a subset of mRNAs are particularly sensitive to inhibition of cap-dependent translation, and that this includes a number of oncogenes such as cyclin D1. We assayed global protein synthesis in MM1.s cells using 35S-methionine and as expected we observed only a modest ≂∼f20% decrease caused by either GNE-652 or GDC-0941 and this decrease was not enhanced by combination treatment. However, we noted across 7 different myeloma cell lines, strong decreases in levels of cyclin D1 that were enhanced by combination treatment. In summary, we have identified several points at which PIM and PI3K/AKT/mTOR converge to provide synergistic apoptosis in multiple myeloma cell lines. These results provide the rationale for further preclinical development of PIM inhibitors and provide the basis for a possible clinical development plan in multiple myeloma. Disclosures: Munugalavadla: Genentech: Employment, Equity Ownership. Berry:Genentech: Employment, Equity Ownership. Chen:Genentech: Employment, Equity Ownership. Deshmukh:Genentech: Employment, Equity Ownership. Drummond:Genentech: Employment, Equity Ownership. Du:Genentech: Employment, Equity Ownership. Eby:Genentech: Employment, Equity Ownership. Fitzgerald:Genentech: Employment, Equity Ownership. S.Friedman:Genentech: Employment, Equity Ownership. E.Gould:Genentech: Employment, Equity Ownership. Kenny:Genentech: Employment, Equity Ownership. Maecker:Genentech: Employment, Equity Ownership. Moffat:Genentech: Employment, Equity Ownership. Moskalenko:Genentech: Employment, Equity Ownership. Pacheco:Genentech: Employment, Equity Ownership. Saadat:Genentech: Employment, Equity Ownership. Slaga:Genentech: Employment, Equity Ownership. Sun:Genentech: Employment, Equity Ownership. Wang:Genentech: Employment, Equity Ownership. Yang:Genentech: Employment, Equity Ownership. Ebens:Genentech Inc: Employment, Equity Ownership.

Download Full-text

DNA Methyltransferase Inhibitors Increase Expression of CD38 and Enhance Daratumumab Efficacy in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-114206 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5618-5618 ◽

Cited By ~ 1

Author(s):

Priya Choudhry ◽

Margarette C. Mariano ◽

Arun P Wiita

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Lines ◽

Research Funding ◽

Plasma Cells ◽

Cpg Island ◽

Ex Vivo ◽

Single Agent ◽

Cd38 Expression

Abstract Introduction: The anti-CD38 monoclonal antibody Daratumumab is highly effective against multiple myeloma, is well tolerated, and has high single agent activity as well as combination effects with lenalidomide-dexamethasone as well as bortezomib-dexamethasone. Patient response to daratumumab monotherapy is highly correlated with pretreatment levels of CD38 expression on MM plasma cells (Nijhof et al, Leukemia (2015) 29:2039) and CD38 loss is correlated with daratumumab resistance (Nijhof et al, Blood (2016) 128:959). As a result, there is significant interest in elucidating the regulation and role of CD38 in MM. Recently, All Trans Retinoic Acid (ATRA), a known small molecule inducer of CD38 in myeloid cells, as well as the FDA-approved histone deacetylase inhibitor panobinostat, were both demonstrated to induce CD38 in MM plasma cells leading to increased lysis by daratumumab. Examining ENCODE data, we found the presence of a CpG island at the first exon of CD38. We hypothesized that removing methylation sites from this CpG island may de-repress CD38 transcription and lead to increased CD38 protein at the cell surface in MM plasma cells. Therefore, here we studied the role of DNA methyl-transferase inhibitors (DNMTis), currently FDA-approved for treatment of myelodysplastic syndrome, as agents to potentiate daratumumab therapy. Methods: We treated MM cell lines (RPMI-8226, MM.1S, XG-1, KMS12-PE) with two different DNMTis, 5-Azacytidine and decitabine, and assessed CD38 cell surface expression by flow cytometry. Similarly, we treated MM patient bone marrow aspirates ex vivo and assessed induction of CD38 expression in the CD138 positive population by flow cytometry. We analyzed CD38 mRNA levels and total CD38 protein levels by qRT-PCR and western blotting respectively. ATRA was used as a positive control in all experiments. We further tested the functional effect of DNMTi treatment on MM cell lines using an Antibody Dependent Cell Cytotoxicity (ADCC) assay. Briefly, live treated cells were incubated overnight with daratumumab and NK92-CD16 transgenic cells at and E:T ratio of 20:1, and lysis was measured using CytoTox-Glo (Promega). Results: Flow analysis revealed that DNMTi treatment induces a 1.2-2 fold increase in CD38 surface protein expression in a dose-dependent manner across MM cell lines. DNMTi treatment consistently yielded similar or higher increases in CD38 expression than that seen in ATRA- or panobinostat-treated cells. Despite significantly lower single-agent cytotoxicity, we found that decitabine led to similar surface CD38 induction as 5-Azacytidine. By RT-qPCR, 5-Azacytidine increased CD38 mRNA expression ~3 fold versus DMSO control, compared to ~2 fold mRNA increase with ATRA. In functional ADCC assays, DNMTi treatment also led to greater lysis than ATRA. Furthermore, the combination of both DNMTi and ATRA was additive, leading to the greatest lysis by NK cells. In contrast, in ex vivo-treated patient samples, ATRA induced greater CD38 expression than 5-Azacytidine on malignant plasma cells. However, this result is expected since MM plasma cells from patients typically do not proliferate in standard ex vivo culture, and active DNA replication is a requirement for successful DNMT inhibition based on known mechanism of action. In patients, however, we anticipate that continual plasma cell proliferation will lead to effective increases in CD38 after DNMTi treatment, as found in MM cell lines here. Summary and Conclusions: Our results here demonstrate that CD38 expression in MM cells is regulated by DNA methylation and targeting DNMTs with small molecule inhibitors leads to increased vulnerability to Daratumumab treatment. We propose that combination treatment with DNMTi and Daratumumab can lead to higher efficacy of daratumumab in daratumumab-naïve MM, as well as reversal of daratumumab-resistance. These combinations should be tested in clinical trials. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

Download Full-text

Adenosine A2A and Beta-2 Adrenergic Receptor Agonist Synergy in B-Cell Malignancies: Selectivity, Breadth of Activity and Effects of Chronic Exposure.

Blood ◽

10.1182/blood.v114.22.3762.3762 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3762-3762 ◽

Cited By ~ 1

Author(s):

Richard J. Rickles ◽

Winnie F. Tam ◽

Antoaneta Necheva ◽

Thomas Giordano ◽

Alexis A. Borisy ◽

...

Keyword(s):

Multiple Myeloma ◽

B Cell ◽

Cell Lines ◽

Adrenergic Receptor ◽

Chronic Exposure ◽

Single Agent ◽

B Cell Malignancies ◽

Cgs 21680 ◽

Beta 2 ◽

Adenosine A2a

Abstract Abstract 3762 Poster Board III-698 By using a high throughput combination screening strategy, we made the surprising discovery that adenosine A2A and beta-2 adrenergic receptor (b2AR) agonists have synergistic anti-proliferative activity in combination with dexamethasone, melphalan, lenalidomide, bortezomib and doxorubicin in preclinical multiple myeloma (MM) models. Both A2A and b2AR agonists are highly selective and demonstrate no single agent activity or synergy in normal cell types including human PBMCs, AoSMCs, HUVECs or HCAECs at concentrations 2-3 orders of magnitude greater than the IC50 in MM cell lines. To further examine the selectivity and breadth we have evaluated A2A and b2AR agonist combinations in a panel of 83 cell lines including solid tumor types and hematological malignancies. Single agents and combinations with dexamethasone and melphalan were systematically studied at multiple ratios of clinically relevant concentrations. Using a quantitative synergy score based on the Loewe model (Lehar et al. Nat Biotech 2009), we observe that combination activity for A2A or b2AR agonists is highly selective for hematologic malignancies with synergy observed most frequently in multiple myeloma and DLBCL cell lines. Synergy is also observed with the B-cell lines JM-1 (pre B-ALL) and GA-10 (Burkitt's lymphoma). Using a relative synergy cut-off (synergy score >1), we find that 13 of the 18 MM cell lines tested demonstrate a synergistic interaction between the A2A agonist CGS-21680 and dexamethasone and 11 demonstrate a synergistic interaction between CGS-21680 and melphalan. Using this same measure, 9 of 18 MM cell lines demonstrate synergy with combinations of the b2AR agonist salmeterol with either dexamethasone or melphalan. Nine and ten of the cell lines in this MM panel are insensitive or respond weakly to dexamethasone and melphalan as single agents respectively. All cell lines were treated with the same concentrations of dexamethasone or melphalan, pointing to A2A agonists having a higher breadth of activity across the MM cell line panel. An interesting observation is the strong synergy observed for A2A or b2AR agonists with dexamethasone in the glucocorticoid-insensitive cell lines EJM and ANBL-6, which suggests that these agents may help restore steroid sensitivity in refractory patients. In general, drug resistance is a recurrent problem for cancer drugs and development of resistance after chronic exposure can reduce drug efficacy and promote refractory disease. We therefore examined the effects of chronic exposure to either A2A or b2AR agonists in the MM.1S cell line. Exposure of cells to CGS-21680 or salmeterol for one month reduced single agent sensitivity >80%. Surprisingly, combinations of either agent with dexamethasone maintained similar amounts of synergy and cell killing as found with naïve untreated cells. As determined by Western blot analysis, the reduction in single agent activity after chronic exposure is not accompanied by a reduction in receptor levels. These data demonstrate that synergistic combinations of A2A and b2AR agonists are highly selective for B-cell malignancies and support the notion that synergistic drug combinations improve therapeutically relevant selectivity and circumvent drug resistance. This work further supports the rationalefor investigation of A2A and b2AR agonists in the treatment of B-cell malignancies and in particular, patients who have MM. Disclosures: Rickles: CombinatoRx, Inc.: Employment. Tam:CombinatoRx, Inc.: Employment. Necheva:CombinatoRx, Inc.: Employment. Giordano:CombinatoRx, Inc.: Employment. Borisy:CombinatoRx, Inc.: Employment. Lee:CombinatoRx, Inc.: Employment.

Download Full-text

Targeting the Wnt/β-Catenin Signaling Pathway and CD44-Mediated Adhesion As a Rational Approach to Overcome Lenalidomide Resistance in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.928.928 ◽

2011 ◽

Vol 118 (21) ◽

pp. 928-928

Author(s):

Chad C. Bjorklund ◽

Sharon Lea Aukerman ◽

Antonia Lopez-Girona ◽

Mohamad Hussein ◽

Rajesh Chopra ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Surface ◽

Signaling Pathway ◽

Cell Lines ◽

Single Agent ◽

Flow Cytometric Analysis ◽

Cellular Adhesion ◽

Rational Approach ◽

Shrna Knockdown

Abstract Abstract 928 Background: Lenalidomide (Len) has been shown to be clinically effective against multiple myeloma (MM) as a single agent and in combination for both newly diagnosed and relapsed/refractory settings, and is increasingly utilized in maintenance therapy. Despite the successful use of Len, some patients display a poor response or become refractory after an initial response. As previously published by our group, hyperactivation of Wnt/β-catenin signaling was identified as a mediating factor for Len-specific resistance in MM cell lines. Additionally, a downstream transcriptional product of Wnt/β-catenin signaling is the extracellular matrix binding protein CD44, which has been implicated in cellular-adhesion-mediated drug-resistance (CAM-DR) in several cancer models, including dexamethasone-resistant MM. These early studies established a rationale to target aberrant Wnt/β-catenin signaling and CD44-mediated CAM-DR to overcome Len resistance. Methods: Wild-type (Wt) and lenalidomide-resistant (LR) MM cell lines were used as models to evaluate the effectiveness of targeting the Wnt/β-catenin signaling pathway and CD44-dependent CAM-DR to overcome Len resistance with Wnt/β-catenin antagonists, shRNA knockdown of CD44, and anti-CD44 neutralization. Results: All-trans-retinoic acid (ATRA) inactivates β-catenin nuclear signaling by altering β-catenin cellular distribution. Combination treatment with Len and ATRA produced a significant (p<0.05) anti-proliferative and cytotoxic effect on LR cells. ATRA exposure of both Wt and LR cells also effectively reduced β-catenin-dependent T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity by as much as 70%, and reversed the apparent increase in activity stimulated by acute Len treatment. Cell surface and total CD44 levels were effectively reduced by as much as 60% when cells were treated with ATRA, even in the presence of Len. Primary patient samples from individuals displaying Len refractory disease were treated ex vivo with Len and ATRA in combination, resulting in a synergistic reduction in cellular viability. We investigated an alternative approach to β-catenin abrogation using FH535, a small molecule β-catenin antagonist that disrupts the transcriptional-dependent association of β-catenin and TCF. Combinations of Len with FH535 resulted in an additive suppression of cellular proliferation on Wt MM cell lines. Similarly, FH535 reduced the viability and enhanced Annexin-V staining on LR KAS-6 and U266 cells by as much as 80% and 55% respectively when used in combination with Len. In addition to selectively targeting β-catenin to overcome LR MM, we evaluated the role of CD44 in LR cell lines. Flow cytometric analysis revealed that cell surface CD44 levels were increased by as much as 11-fold in LR KAS-6 cells when compared to Wt cells. Acute treatment of Wt ANBL-6 and MM1.S cells with Len moderately enhanced the cell surface levels of CD44 by approximately 1.5-fold. The enhanced CD44 surface levels induced by Len on LR ANBL-6 and KAS-6 corresponded to at least a 2-fold increase in cellular adhesion to bone marrow stromal cells, and enhanced adhesion to increasing concentrations of hyaluranon (HA). Pre-incubation with free HA or CD44 neutralizing antibodies reduced the adhesive properties of both Wt and LR ANBL-6 and KAS-6 cell lines. shRNA knockdown of CD44 sensitized LR cells to Len when compared to a non-specific scrambled shRNA transfection. Interestingly, extensive statistical analysis of banked gene expression data from primary samples located at the Multiple Myeloma Research Consortium (MMRC) database revealed a significant correlation (p=0.01) between high expression levels of CD44 and a poor outcome prognosis, albeit irrespective of treatment regimens. Furthermore, current analysis of surface CD44 levels on primary MM bone marrow aspirates suggests that those patients who have received prior Len therapy have an overall increase in CD44 when compared to Len naïve patients. Conclusions: Collectively, our results suggest that MM cell Len resistance is, at least in part, dependent on β-catenin and CD44 and that selective targeting of these cellular proteins in conjunction with Len treatment represents a rational approach for clinical treatment of Len-resistant MM. Disclosures: Aukerman: Celgene Corporation: Employment. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Hussein:Celgene Corporation: Employment. Chopra:Celgene Corporation: Employment. Orlowski:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Download Full-text

Systems Biology Analysis Identifies Targetable Vulnerability Networks to Proteasome Inhibitors in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-120269 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 950-950

Author(s):

Mark B. Meads ◽

Rafael Renatino Canevarolo ◽

Praneeth Reddy Sudalagunta ◽

Paula S. Oliveira ◽

Dario M. Magaletti ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Cell Line ◽

Cell Lines ◽

Ex Vivo ◽

Single Agent ◽

Proteasome Inhibitors ◽

In Vivo Models ◽

Viability Assay

Abstract Proteasome inhibitors (PI) such as bortezomib and carfilzomib are critical components of anti-multiple myeloma (MM) therapy, yet all MM patients eventually develop refractory disease. We developed a non-biased method to identify and validate dysregulated pathways associated with PI-resistance in myeloma by combining RNAseq data from 522 MM patient specimens obtained from our Total Cancer Care/M2Gen/ORIEN network at Moffitt Cancer Center with paired ex vivo sensitivity to PIs and kinase inhibitors (KI). Dimensionality reduction analysis (t-SNE) and Fuzzy C-means was used to identify 422 clusters of genes that co-express in individual patients, and Gene Set Enrichment Analysis (GSEA) was used to identify clusters with gene expression patterns that correlated with PI sensitivity. Using publicly curated databases and in silico integrative analyses, we built protein-protein interaction networks to identify putative transcription factors, corresponding master regulators (kinases), and candidate KIs to promote PI sensitization. This systems biology approach identified a Chk1-Cdk1-Plk1 circuit associated with PI-resistance and also found 21 additional kinases (of 501 expressed in our cohort's kinome) that could be targeted to re-sensitize PI-resistant MM, which we confirmed in cell lines, specimens from relapsed patients, and two in vivo models. A panel of paired isogenic PI-resistant and sensitive MM cell lines were differentially screened to find kinases associated with PI-resistance using activity-based protein profiling (ABPP) and KI activity measured by high-throughput viability assay. The MM cell lines 8226 and U266, along with their drug resistant counterparts 8226-B25 and U266-PR, were grown in mono-culture for 24h and lysates were enriched for ATP binding proteins by affinity purification versus a chemical probe. Tryptic peptides were measured using discovery proteomics (nano-UPLC and QExactive Plus mass spectrometer) to identify 85 kinases out of a total of 715 proteins in 8226-B25 MM cells and 35 kinases out of a total of 688 proteins in U266-PR MM cells that were preferentially enriched by 2-fold change compared to parental cell lines. Twenty-four kinases were commonly activated among PI-resistant cell line pairs and were screened in PI-resistant myeloma lines using a label-free, high throughput viability assay that simulates the tumor microenvironment. Three KIs targeting Plk1 (volasertib and GSK461364) and Cdk1/5 (dinaciclib) consistently maintained LD50s in the low-nanomolar range and induced caspase-3 activation in four PI-resistant MM cell lines: 8226-B25, U266-PR, ANBL-6-V10R, and Kas6-V10R. Twenty-four kinases each were identified by RNAseq/ex vivo PI sensitivity of MM specimens and ABPP of PI-resistant/sensitive MM cell line pairs. Of these, 7 kinases were identified by both methods: Cdk1, Chk1, Plk1, ILK, Syk, PKA, and p70S6K. Several KIs targeting Cdk1, Plk1, ILK, DNAPK, Syk, MKK7, Nek2, and mTOR identified in patient specimen or cell-line screens showed single agent activity in MM patient bone marrow specimens purified by a CD138 affinity column. Among these, inhibitors to Cdk1, ILK, mTOR, and Plk1 showed the most activity in patient specimens with an average 96h LD50 of 25 nM (n=56), 2.4 uM (n=42), 2.7 uM (n=57) and 3.8 uM (n=53), respectively. Six KIs targeting Plk1, ILK, Syk, MKK7, Nek2 and MARK3 were synergistic with carfilzomib in 20 patient specimens and maintained or improved ex vivo activity in relapsed refractory MM (RRMM) specimens. Volasertib, which targets Plk1, was the most synergistic with carfilzomib of all KIs tested in patient specimens and was further validated in two in vivo models: a NSG/U266 xenograft model of PI resistance and the syngeneic C57BL/6-KaLwRij/5TGM1 immunocompetent model. Volasertib significantly increased survival and reduced tumor burden in both models as a single agent, and was more effective versus PI-resistant tumors compared to PI-sensitive counterparts. Our pharmaco-proteomic screen, coupled with rich gene expression data from patients identified Plk1 as a target critical to MM survival in the context of acquired PI resistance and represents a unique workflow to find tumor vulnerabilities that arise during therapy. We anticipate that these data will also produce a critical path for the personalized allocation of therapy to maximize efficacy and minimize the use of ineffective therapies in RRMM. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Biologic sequelae of IκB kinase (IKK) inhibition in multiple myeloma: therapeutic implications

Blood ◽

10.1182/blood-2008-06-161505 ◽

2009 ◽

Vol 113 (21) ◽

pp. 5228-5236 ◽

Cited By ~ 57

Author(s):

Teru Hideshima ◽

Dharminder Chauhan ◽

Tanyel Kiziltepe ◽

Hiroshi Ikeda ◽

Yutaka Okawa ◽

...

Keyword(s):

Multiple Myeloma ◽

Growth Inhibition ◽

Cell Lines ◽

Nuclear Factor Κb ◽

Down Regulation ◽

Growth And Survival ◽

Therapeutic Implications ◽

Iκb Kinase ◽

Canonical Pathways ◽

Promising Treatment

Abstract Nuclear factor-κB (NF-κB) has an important role in multiple myeloma (MM) cell pathogenesis in the context of the bone marrow (BM) microenvironment. In NF-κB signaling cascades, IκB kinase α (IKKα) and IKKβ are key molecules that predominantly mediate noncanonical and canonical pathways, respectively. In this study, we examined the biologic sequelae of the inhibition of IKKα versus IKKβ in MM cell lines. All MM cell lines have constitutive canonical NF-κB activity, and a subset of MM cell lines shows noncanonical NF-κB activity. Adhesion to BM stromal cells further activates both canonical and noncanonical NF-κB activity. IKKβ inhibitor MLN120B blocks canonical pathway and growth of MM cell lines but does not inhibit the noncanonical NF-κB pathway. Although IKKα knockdown induces significant growth inhibition in the cell lines with both canonical and noncanonical pathways, it does not inhibit NF-κB activation. Importantly, IKKα down-regulation decreases expression of β-catenin and aurora-A, which are known to mediate MM cell growth and survival. Finally, IKKβ inhibitor enhances the growth inhibition triggered by IKKα down-regulation in MM cells with both canonical and noncanonical NF-κB activity. Combination therapy targeting these kinases therefore represents a promising treatment strategy in MM.

Download Full-text

AR-42, a novel HDAC inhibitor, exhibits biologic activity against malignant mast cell lines via down-regulation of constitutively activated Kit

Blood ◽

10.1182/blood-2009-07-231985 ◽

2010 ◽

Vol 115 (21) ◽

pp. 4217-4225 ◽

Cited By ~ 53

Author(s):

Tzu-Yin Lin ◽

Joelle Fenger ◽

Sridhar Murahari ◽

Misty D. Bear ◽

Samuel K. Kulp ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Cell Lines ◽

Histone Deacetylase Inhibitors ◽

Ex Vivo ◽

Histone Deacetylation ◽

Down Regulation ◽

Biologic Activity ◽

Promising Therapeutic Approach

Histone hypoacetylation occurs in many cancers and inhibition of histone deacetylation is a promising approach to modulate these epigenetic changes. Our laboratory previously demonstrated that the histone deacetylase inhibitors (HDACis) vorinostat and AR-42 reduced the viability of a canine malignant mast cell line. The purpose of this study was to further investigate the mechanisms of pan-HDAC inhibition in normal and malignant mast cells. Mouse and canine malignant mast cell lines expressing various Kit mutations, normal canine mast cells, and primary canine malignant mast cells were treated with AR-42 (a novel HDACi) and effects on cell viability, cycling, and signaling were evaluated. Treatment with AR-42 induced growth inhibition, cell- cycle arrest, apoptosis, and activation of caspases-3/7. AR-42 promoted hyperacetylation of H3, H4, and alpha-tubulin, and up-regulation of p21. Down-regulation of Kit occurred after AR-42 treatment via inhibition of Kit transcription. Disassociation between Kit and heat shock protein 90 (HSP90) and up-regulation of HSP70 were observed after AR-42 treatment, suggesting potential loss of HSP90 chaperone function. Lastly, AR-42 down-regulated the expression of p-Akt, total Akt, phosphorylated STAT3/5 (pSTAT3/5), and total STAT3/5. In summary, AR-42 exhibits in vitro and ex vivo biologic activity against malignant mast cells, representing a promising therapeutic approach for malignant mast cell disease.

Download Full-text

Activity of Tyrosine Kinase Inhibitors in Multiple Myeloma.

Blood ◽

10.1182/blood.v110.11.4804.4804 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4804-4804

Author(s):

Reinhold Munker ◽

Cory Cordova ◽

Paula Polk ◽

Charles V. Wendling ◽

Amanda W. Sun ◽

...

Keyword(s):

Multiple Myeloma ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Cell Lines ◽

Kinase Inhibitors ◽

Single Agent ◽

Calf Serum ◽

Gene Expression Pattern ◽

Inhibition Of Proliferation ◽

Short Term Exposure

Abstract Tyrosine kinase inhibitors (TKIs) have been successfully introduced for the treatment of cancer. Imatinib, dasatinib and nilotinib target bcr/abl and were found to induce molecular remissions in chronic myeloid leukemia. Imatinib has also been found to be active in other malignancies like gastrointestinal stromal tumors. Sunitinib and sorafenib are multi-targeted tyrosine kinase inhibitors and so far, have shown activity against renal cell carcinoma and other cancers. Gefitinib targets the tyrosine kinase of epidermal growth factor receptor and has been found to be active against some cases of non-small cell lung cancer. There is circumstantial evidence that tyrosine kinases and their receptors (e.g. VEGF, IGF-1 and FGFR3) are active in multiple myeloma. In order to develop new treatments for multiple myeloma (MM), we tested several currently available TKIs for their activity against MM cell lines. Materials and methods: The a cell lines MC/CAR, ARH77, RPMI 8226, ARP1, JJN3, MM1S, and INA-6 were treated with various concentrations of TKIs and analyzed for cell growth in liquid culture, proliferation, apoptosis, and gene expression pattern screening 14,500 genes using U133A_2 arrays. Results: Imatinib, nilotinib, dasatinib, gefitinib induced cytotoxicity in most cases at high concentrations (50% inhibitory concentration ≥ 100 μMol), whereas sunitinib and sorafenib were active at lower concentrations (50% IC 1– 5 μMol). The cytoxicity was observed early (within 4 to 24 hours of exposure) and involves apoptosis. Interleukin-6 did not offer protection against the cytotoxicity of sorafenib or sunitinib, however the inhibition of proliferation was more pronounced in low fetal calf serum (2.5 versus 10%). A short-term exposure of the myeloma cell line MM1S to 10 μMol sorafenib resulted in more than 2 fold changes in 283 genes or sequences (175 up, 108 down). If only 10 fold changes are considered, 21 genes or sequences were upregulated (mainly enzymes, regulators and ligands) and 11 downregulated (mainly regulatory proteins, among them IL6 signal transducer). Conclusion: We found that the multitargeted TKIs sorafenib and sunitinib are active in vitro against multiple myeloma. We plan to investigate patient samples, and to elucidate the targets and the mechanisms of action. Our data will support clinical trials both as single agent and in combination with other drugs like bortezomib, thalidomide, alkylators and ionizing radiation.

Download Full-text