Synergistic Effects of Venetoclax and Daratumumab on Antibody-Dependent Cell-Mediated Natural Killer Cytotoxicity in Multiple Myeloma

The prognosis of multiple myeloma (MM) has drastically improved owing to the development of new drugs, such as proteasome inhibitors and immunomodulatory drugs. Nevertheless, MM is an extremely challenging disease, and many patients are still refractory to the existing therapies, thus requiring new treatment alternatives. Venetoclax is a selective, orally bioavailable inhibitor of BCL-2 that shows efficacy in MM not only as a single agent but also in combination therapy, especially for MM patients with translocation t(11;14). However, many patients are refractory to this drug. Here, we treated the MM cell lines KMS12PE and KMS27 with a combination treatment of venetoclax targeting BCL-2 and daratumumab targeting CD38 to evaluate the synergistic cytotoxicity of these drugs in vitro. MM cell lines were co-cultured with natural killer (NK) cells at an effector:target ratio of 0.3:1 in the presence of serial concentrations of daratumumab and venetoclax, and the resulting apoptotic MM cells were detected by flow cytometry using annexin V. These results indicated that the antibody-dependent cell-mediated NK cytotoxicity was enhanced in KMS12PE and KMS27 cells harboring t(11;14) with a high BCL-2 expression, suggesting that the combination treatment of venetoclax and daratumumab should be especially effective in patients with these characteristics.

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Efficacy of Panobinostat (LBH589) in Multiple Myeloma Cell Lines and In Vivo Mouse Model: Tumor-Specific Cytotoxicity and Protection of Bone Integrity in Multiple Myeloma.

Blood ◽

10.1182/blood.v110.11.1510.1510 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1510-1510 ◽

Cited By ~ 2

Author(s):

Joseph D. Growney ◽

Peter Atadja ◽

Wenlin Shao ◽

Youzhen Wang ◽

Minying Pu ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Bone Density ◽

Mouse Model ◽

Cortical Bone ◽

Cell Lines ◽

Single Agent ◽

Synergistic Cytotoxicity

Abstract Panobinostat (LBH589) is a highly potent oral pan-deacetylase (DAC) inhibitor currently undergoing clinical development in hematologic and solid malignancies. Here we report the effects of panobinostat on multiple myeloma (MM) cells in vitro and in a murine xenograft model in vivo. Panobinostat exhibited potent cytotoxic activity (IC50 <10 nM) against 8 MM cell lines (KMS-12PE, KMS-18, LP-1, NCI H929, KMS-11, RPMI8226, OPM-2, and U266). Panobinostat has been shown to affect signals involved in MM cell-cycle arrest and cell death, and to induce apoptosis via mitochondrial perturbation. In addition, panobinostat has been shown to selectively induce cell death of plasma cells isolated from MM patients without toxicity to normal lymphocytes or granulocytes. To investigate the effect of panobinostat in vivo, a disseminated luciferized MM.1S xenograft mouse model was treated with vehicle or panobinostat 15 mg/kg by intraperitoneal (i.p.) administration qd×5 for 3 weeks. Panobinostat treatment reduced the burden of MM.1S tumor cells to 22% treated over control (T/C) relative to vehicle-treated animals. In addition, MM.1S tumor-bearing mice treated with panobinostat displayed reduced trabecular and cortical bone damage relative to vehicle-treated animals. The mean ± SEM trabecular bone density and cortical bone density (% Bone Volume/Total Volume) of panobinostat-treated animals was 14.5% ± 2.0 and 98.1% ± 0.4, respectively, compared with 2.2% ± 0.3 and 89.1% ± 1.5 in vehicle-treated animals. In combination with the proteosome inhibitor bortezomib (BZ), panobinostat displayed significant synergistic cytotoxicity without additional toxicity to normal bone marrow stromal cells in vitro. In the MM.1S-luciferase tumor mouse model, combined treatment with panobinostat at 10 mg/kg i.p. qd×5 for 4 weeks and BZ at 0.2 mg/kg intravenously 1qw for 4 weeks reduced tumor burden to 7% T/C relative to vehicle, panobinostat alone (31% T/C), or BZ alone (44% T/C). Disease progression, measured as median time to endpoint (TTE) was improved from 37 to 54 days (P<0.05) by panobinostat and to 46 days by BZ (P<0.05). The combination treatment further improved clinical outcome relative to both single-agent treatment groups (P<0.05), extending the TTE to 73 days. In contrast to BZ, the immunomodulatory drug thalidomide (TH) had no significant single-agent activity at 150 mg/kg p.o. qd for 4 weeks. However, combination activity (18% T/C) was observed when TH was combined with a sub-efficacious dose of panobinostat (5 mg/kg, 64% T/C). Combination of panobinostat and TH increased the TTE to 50 days, compared with 37.5, 43, and 39.5 days (P<0.05), respectively, for the vehicle, panobinostat, or TH as single agents. These data demonstrate that panobinostat exhibits significant anti-proliferative and anti-tumor activities on MM cells both in vitro and in vivo. Panobinostat, as a single agent or in combination with BZ or TH, is a promising therapy for MM, and these studies may provide the rationale for clinical evaluation of panobinostat and BZ combination in the treatment of MM.

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Concurrent Inhibition of Pim and Akt Pathways with Pim447 and Afuresertib Activates FOXO3a and Depletes c-Myc to Induce Synergistic Cell Death in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.3007.3007 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3007-3007

Author(s):

Niamh Keane ◽

Mairead Reidy ◽

Alessandro Natoni ◽

Michael O'Dwyer

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Death ◽

Cyclin D1 ◽

Cell Lines ◽

Combination Treatment ◽

Single Treatment ◽

Synergistic Cytotoxicity ◽

Inhibitor Treatment

Abstract Background Dual targeting of overlapping pathways in hematologic malignancies is an attractive therapeutic strategy to prevent resistance by compensatory signalling. The Pim and PI3K/AKT/mTOR pathways are proving effective drug targets in Multiple Myeloma (MM). Pim inhibition with novel compound Pim447 has demonstrated durable single agent activity in advanced disease (Raab et al ASH 2014, NCT01456689) while Akt inhibitor Afuresertib is effective in relapsed MM in combination with bortezomib (Voorhees et al ASH 2013, NCT01428492). Pim and Akt converge on mTOR activation, inhibition of p53, and the inactivation of BAD via phosphorylation. These redundant roles suggest potential for compensatory signalling on single pathway blockade. We have hence evaluated these clinically relevant drugs to provide a preclinical rationale for their combination in MM. Methods Pim447 and Afuresertib were evaluated against a panel of MM cell lines - NCI-H929, RPMI-8226, MM1S and KMS11 - and primary MM bone marrow aspirate samples. Cell Viability (AnnexinV/PI) and Cell Cycle (Edu/DAPI) analysis was performed up to 72 hours. Compusyn software was used for Chou-Talalay generated combination indices (CI <1 indicating synergy). Results Single treatment with either Pim447 or Afuresertib reduces proliferation and induces G1 accumulation but is not cytotoxic to MM cells. We tested 25 Pim447 and Afuresertib combinations against each cell line and found combinations of nanomolar concentrations result in >90% cell death at 48hours (CI 0.1-0.8) (Figure 1). For analysis of mechanism of synergy we selected the lowest synergistic combinations at which there is evidence of both drug/target engagement (i.e. hyperphosphorylation of Akt by Afuresertib and increase in Pim-2 by Pim447) and activity against validated downstream targets (FOXO3a for Afuresertib and BAD for Pim447). Interestingly, Pim inhibitor treatment leads to an increase in active phospho-Akt and phosphorylation of its target FOXO3a. Conversely, with Akt inhibition we demonstrate increased Pim-2 expression above basal levels, providing evidence of compensatory signalling on single target inhibition. In keeping with this, dual treatment results in greater inhibition of overlapping targets mTOR (measured via reduced outputs phospho-S6 and phospho-4EBP1), and phospho-BAD. Combination treatment uniquely depletes c-Myc in all cell lines, with single drugs having no effect (Figure 2 NCI-H929). Consistent with this, while neither inhibitor alone reduces phosphorylation of Akt target GSK3β the combination does so effectively, thus activating GSK3β which in turn can target c-Myc for ubiquitination. Further, with activation of GSK3β on dual blockade we demonstrate a concerted reduction in Cyclin D1. As noted above, Akt-induced (inactivating) phosphorylation of transcription factor FOXO3a is predictably inhibited by Afuresertib treatment. Unexpectedly active FOXO3a was upregulated on combination treatment and may mediate synergistic cytotoxicity via derepression of target genes. Pim447 and Afuresertib combination markedly potentiates G1 cell cycle arrest. This does not occur by induction of shared targets p53 or p21, but is associated with induction of FOXO3a transcriptional target p15. Further, in 3 of 4 cell lines the FOXO3a target p27 is increased to a greater extent by dual inhibitor treatment, consistent with its transcription on loss of repressor function of c-Myc. Co-culture with HS5 stromal cells affords protection against Afuresertib-mediated cell death in vitro, however, this is overcome and synergistic cell death observed with combination treatment. We have tested the combination in 10 primary MM samples to date. The inhibitors were active in all samples and the combination increased cell death in 6/10 (Figure 3). Conclusions Single treatment with Pim447 or Afuresertib is not cytotoxic in vitro at clinically relevant concentrations. However, combined inhibition activates GSK3β with reduction in Cyclin D1 and depletion of c-Myc, and induces transcriptional regulator FOXO3a resulting in impressive synergistic cell death. Ongoing preclinical assessment of this promising combination will include testing in the VK*MYC transgenic mouse model. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures No relevant conflicts of interest to declare.

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Anticancer effects of the combined Thai noni juice ethanolic extracts and 5-fluorouracil against cholangiocarcinoma cells in vitro and in vivo

Scientific Reports ◽

10.1038/s41598-021-94049-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jeerati Prompipak ◽

Thanaset Senawong ◽

Banchob Sripa ◽

Albert J. Ketterman ◽

Suppawit Utaiwat ◽

...

Keyword(s):

Nude Mice ◽

Combination Treatment ◽

Synergistic Effects ◽

Single Agent ◽

Xenograft Model ◽

Ethanolic Extract ◽

Model Combination ◽

Mouse Xenograft Model ◽

Ethanolic Extracts

AbstractApplication of 5-fluorouracil (5-FU) in cholangiocarcinoma (CCA) is limited by adverse side effects and chemoresistance. Therefore, the combination therapy of 5-FU with other substances, especially natural products may provide a new strategy for CCA treatment. The aim of this study was to evaluate the combination effects of 5-FU and two ethanolic extracts of Thai noni juice (TNJ) products on CCA cell lines and nude mice xenografts. The results of antiproliferative assay showed the combination treatment of 5-FU and each TNJ ethanolic extract exerted more cytotoxicity on CCA cells than either single agent treatment. Synergistic effects of drug combinations can enable the dose reduction of 5-FU. The mechanism underlying a combination treatment was apoptosis induction through an activation of p53 and Bax proteins. In the nude mouse xenograft model, combination treatments of 5-FU with each TNJ ethanolic extract suppressed the growth of CCA cells implanted mice more than single agent treatments with no effects on mouse body weight, kidney, and spleen. Moreover, low doses of TNJ ethanolic extracts reduced the hepatotoxicity of 5-FU in nude mice. Taken together, these data suggested that the ethanolic extracts of TNJ products can enhance the anti-CCA effect and reduce toxicity of 5-FU.

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PL3.6 Targeting GSK-3 activity promotes mitotic catastrophe via centrosome destabilisation and enhances the effect of radiotherapy in glioma models

Neuro-Oncology ◽

10.1093/neuonc/noz126.009 ◽

2019 ◽

Vol 21 (Supplement_3) ◽

pp. iii4-iii4

Author(s):

A Bruning-Richardson ◽

H Sanganee ◽

S Barry ◽

D Tams ◽

T Brend ◽

...

Keyword(s):

Cell Lines ◽

Cancer Progression ◽

Combination Treatment ◽

Single Agent ◽

Drug Penetration ◽

In Vivo Models ◽

Human Astrocytes ◽

Normal Human

Abstract BACKGROUND Targeting kinases as regulators of cellular processes that drive cancer progression is a promising approach to improve patient outcome in GBM management. The glycogen synthase kinase 3 (GSK-3) plays a role in cancer progression and is known for its pro-proliferative activity in gliomas. The anti-proliferative and cytotoxic effects of the GSK-3 inhibitor AZD2858 were assessed in relevant in vitro and in vivo glioma models to confirm GSK-3 as a suitable target for improved single agent or combination treatments. MATERIAL AND METHODS The immortalised cell line U251 and the patient derived cell lines GBM1 and GBM4 were used in in vitro studies including MTT, clonogenic survival, live cell imaging, immunofluorescence microscopy and flow cytometry to assess the cytotoxic and anti-proliferative effects of AZD2858. Observed anti-proliferative effects were investigated by microarray technology for the identification of target genes with known roles in cell proliferation. Clinical relevance of targeting GSK-3 with the inhibitor either for single agent or combination treatment strategies was determined by subcutaneous and orthotopic in vivo modelling. Whole mount mass spectroscopy was used to confirm drug penetration in orthotopic tumour models. RESULTS AZD2858 was cytotoxic at low micromolar concentrations and at sub-micromolar concentrations (0.01 - 1.0 μM) induced mitotic defects in all cell lines examined. Prolonged mitosis, centrosome disruption/duplication and cytokinetic failure leading to cell death featured prominently among the cell lines concomitant with an observed S-phase arrest. No cytotoxic or anti-proliferative effect was observed in normal human astrocytes. Analysis of the RNA microarray screen of AZD2858 treated glioma cells revealed the dysregulation of mitosis-associated genes including ASPM and PRC1, encoding proteins with known roles in cytokinesis. The anti-proliferative and cytotoxic effect of AZD2858 was also confirmed in both subcutaneous and orthotopic in vivo models. In addition, combination treatment with AZD2858 enhanced clinically relevant radiation doses leading to reduced tumour volume and improved survival in orthotopic in vivo models. CONCLUSION GSK-3 inhibition with the small molecule inhibitor AZD2858 led to cell death in glioma stem cells preventing normal centrosome function and promoting mitotic failure. Normal human astrocytes were not affected by treatment with the inhibitor at submicromolar concentrations. Drug penetration was observed alongside an enhanced effect of clinical radiotherapy doses in vivo. The reported aberrant centrosomal duplication may be a direct consequence of failed cytokinesis suggesting a role of GSK-3 in regulation of mitosis in glioma. GSK-3 is a promising target for combination treatment with radiation in GBM management and plays a role in mitosis-associated events in glioma biology.

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A Phase II Study of PXD101 in Advanced Multiple Myeloma.

Blood ◽

10.1182/blood.v108.11.3583.3583 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3583-3583 ◽

Cited By ~ 11

Author(s):

Daniel Sullivan ◽

Seema Singhal ◽

Michael Schuster ◽

James Berenson ◽

Peter Gimsing ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Stable Disease ◽

Progressive Disease ◽

Synergistic Effects ◽

Objective Response ◽

Study Drug ◽

Primary Objective

Abstract Background: PXD101 is a small molecule HDAC inhibitor of the hydroxamate class, which demonstrates broad anti-neoplastic activity in vitro and in vivo. PXD101 has antiproliferative activity on multiple myeloma cell lines, and shows additive/synergistic effects with standard agents used in myeloma, against these cell lines. PXD101 is being tested as monotherapy and in combination with standard agents for treatment of multiple myeloma. Methods: The primary objective of this study was to assess the activity of PXD101 alone or with dexamethasone, in multiple myeloma patients (pts) who have failed at least 2 prior therapies. Response was measured using the Blade criteria. PXD101 was administered as a 30-min IV infusion on Days 1–5 of a 3-wk cycle, at a dose of 1000 mg/m2/d (900 mg/m2/d in earlier patients). Patients are initially treated with PXD101 alone for two cycles. At the end of cycle two and every cycle thereafter, pts are evaluated for tumor response and continue on the study as follows: pts with objective response or stable disease continue on PXD101 monotherapy, while pts who have progressive disease (PD) are treated with a combination of PXD101 + dexamethasone (Dex). Dex was given orally 40 mg daily on Days 2–5 and 10–13 of the treatment cycle. Results: To date, 24 pts have been enrolled, 19 for which data are currently available. These pts have received a median of 5 (range 2–10) prior therapies. Seventeen pts are evaluable, 12 of whom are evaluable for ≥ 2 cycles, and 5 evaluable for 1 cycle only; 2 pts are unevaluable due to inconsistent baseline that prevented response assessment. Of the 5 pts evaluable for 1 cycle only, 4 discontinued due to PD and one withdrew from study. The 12 pts evaluable for ≥ 2 cycles received a median of 4 treatment cycles (range 2–12); 6 of these patients went on to receive PXD101+Dex. In these 12 pts, duration of PXD101 monotherapy was for 2–4 cycles, with almost all pts (10) receiving only 2 cycles. PXD101+Dex treatment in 6 pts was for 1–10 cycles (10, 6, 4, 4, 3, and 1). In 12 pts on monotherapy for ≥ 2 cycles, there were 6 SD (duration 6–12 wks) and 6 PD. The short duration of SD in PXD101 monotherapy was attributed to patient withdrawal or moving to Dex addition in spite of disease stabilization. All 6 pts receiving PXD101+Dex had previously received at least 2 Dex-containing regimens. One pt had MR (duration 6 wks), and 5 pts had SD. One pt has had SD for 35 wks, with 90% decrease in serum M-component sustained in the last 12 wks; another pt has had SD for 15 wks. In 69 cycles of treatment there were 7 Grade 3/4 adverse events assessed by the investigator as potentially related to study drug. These include anemia (2), infection, respiratory distress, hyperglycemia, thrombocytopenia, and fatigue. Conclusions: PXD101 treatment has resulted in stabilization of advanced and progressive disease, providing clinical benefit to patients. PXD101 combination with dexamethasone led to an MR as well as long duration of stable disease in patients who have previously received multiple Dex regimens. These observations support the continued exploration of PXD101 in combination with other agents for treatment of multiple myeloma.

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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.

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A Critical Role for PIM2 Kinase in Multiple Myeloma Through NF-κB Activation

Blood ◽

10.1182/blood.v118.21.1839.1839 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1839-1839

Author(s):

Veerendra Munugalavadla ◽

Leanne Berry ◽

Jae Chang ◽

Geoffrey Del Rosario ◽

Jake Drummond ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Cyclin D1 ◽

Cell Lines ◽

Combination Treatment ◽

Single Agent ◽

Myeloma Cell ◽

Myeloma Cells ◽

Pim Kinases ◽

Pi3k Inhibition

Abstract Abstract 1839 The PIM kinases are a family of 3 growth factor- & cytokine-induced proteins hypothesized to have redundant survival and growth functions. Although PIM-1, -2 have been noted as highly expressed in multiple myeloma (MM) (Claudio JO et al., 2002), there are few data to support potential therapeutic utility of PIM inhibition in this indication. Here we show that the myeloma cell lines express all PIM protein isoforms to varying extents, and we describe the properties of a novel pan-PIM inhibitor GNE-652 with picomolar biochemical potency, an excellent selectivity profile, and favorable ADME properties. Myeloma cell lines and patient samples exhibit a striking prevalence of response to GNE-652 (23 of 25 lines with IC50 < 1 micromolar, median < 0.1 micromolar) and synergy in combination with the PI3K inhibitor GDC-0941 (mean combination index values ∼0.2 (n=25)). MM cells respond to this combination with cell cycle arrest and marked apoptosis in vitro. Conversely, a PIM-1, -3 selective inhibitor, GNE-568, failed to suppress MM cell growth and also failed to provide synergy in combination with PI3K inhibition, suggesting PIM-2 is a critical driver of MM cell growth & survival. Additional results suggest that PIM signaling converges on both TORC1 and AKT to generate differential synergies with PI3K/AKT/mTOR pathway inhibitors. 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 resulted 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. 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 all the myeloma cell lines tested. 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 × IC50 concentrations for single agents, combination drug treatment achieved greater extent of effect than single agent treatment. It has been hypothesized that a subset of mRNAs are particularly sensitive to inhibition of cap-dependent translation, including a number of oncogenes such as cyclin D1. We noted across 7 different myeloma cell lines, strong decreases in levels of cyclin D1, and D3 that were further decreased by combination treatment of PIM and PI3K inhibition. In summary, we have identified several points at which PIM and PI3K/AKT/mTOR converge to provide synergy in multiple myeloma cell lines. As PIM isoforms are highly expressed in MM cells, we hypothesized that this could be due to proteosomal-mediated stability, and interestingly, MG132 and velcade each stabilized all PIM isoforms. It is commonly known that the JAK/STAT pathway regulates PIM transcription, but we show JAK inhibitors failed to abolish the expression of PIM in myeloma cells, suggesting a role for additional regulators. Recent genome sequencing studies from human myeloma samples (Chapman MA et al., 2011) confirmed the prevalence of NF-kB pathway activation, consistent with prior observations made in MM cell lines (Demchenko YN et al., 2010). The relationship of PIM and NF-kB is controversial in the literature (Hammerman PS et al., 2004 & Zhu N et al., 2002), with some groups placing PIM upstream of NF-kB and others the converse. Using an IκBα inhibitor, BMS-345541, we have examined the role for NF-kB in the regulation of PIM kinases. Here, we show that the BMS-345541 could preferentially suppress PIM2 expression in a dose dependent manner while PIM 1, 3 levels are modestly affected, suggesting that the high levels of PIM2 expression observed are partly driven by deregulation of the NF-kB pathway in MM. In conclusion, we provide pharmacological and biochemical evidence to suggest that PIM2 differentially regulate growth and survival of myeloma cells. Our results provide the rationale for further preclinical development of PIM inhibitors and the basis for a possible clinical development plan in multiple myeloma. Disclosures: Munugalavadla: Genentech: Employment. Berry:Genentech: Employment. Chang:Genentech: Employment. Rosario:Genentech: Employment. Drummond:Genentech: Employment. Du:Genentech: Employment. Fitzgerald:Genentech: Employment. Friedman:Genentech: Employment. Gould:Genentech: Employment. Maecker:Genentech: Employment. Moffat:Genentech: Employment. Slaga:Genentech: Employment. Xiaojing:Genentech: Employment. West:Genentech: Employment. Yu:Genentech: Employment. Ebens:Genentech: Employment.

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EZH2 Inhibitors Are Broadly Efficacious in Multiple Myeloma As Single Agent and in Combination with Standard of Care Therapeutics

Blood ◽

10.1182/blood.v128.22.5672.5672 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5672-5672 ◽

Cited By ~ 1

Author(s):

Shilpi Arora ◽

Kaylyn Williamson ◽

Shruti Apte ◽

Srividya Balachander ◽

Jennifer Busby ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Single Agent ◽

Standard Of Care ◽

Tumor Growth Inhibition ◽

Sequencing Data ◽

Employment Equity ◽

Chip Sequencing ◽

Equity Ownership

Abstract Post-translational modifications of the histone proteins play a key role in regulating processes that require access to DNA. Specifically, methylation of lysine 27 on histone 3 (H3K27) is intimately linked with transcriptional repression. EZH2, a histone lysine methyl transferase is the catalytic component of the PRC2 complex, which catalyzes H3K27 methylation. EZH2 dysregulation has been observed in different malignancies and inhibition of its catalytic activity has emerged as a novel therapeutic approach to treat human cancers. Potent, selective and reversible EZH2 small molecule inhibitors are currently being tested in Ph. 1 clinical trials. We and others have reported EZH2 dependencies across non-Hodgkin Lymphoma subtypes in cancer cell lines, in xenograft mouse models and in lymphoma patients. We identified Multiple Myeloma as potential clinical application for EZH2 inhibitors. Treatment with EZH2 inhibitors such as CPI-360, CPI-169 and CPI-1205 cause apoptosis in multiple myeloma and plasmacytoma cell models and causes tumor growth inhibition in myeloma xenograft models at well tolerated doses. An EZH2-controlled transcriptional signature across various multiple myeloma was identified using integrated RNA-sequencing and ChIP-sequencing data. Combination studies testing EZH2 inhibitors with standard of care (SOC) agents across a panel of multiple myeloma cell lines showed synergistic responses with several of the SOC agents in vitro and in vivo. Disclosures Arora: Constellation Pharmaceuticals: Employment, Equity Ownership. Williamson:Constellation Pharmaceuticals: Employment, Equity Ownership. Apte:Constellation Pharmaceuticals: Employment, Equity Ownership. Balachander:Constellation Pharmaceuticals: Employment, Equity Ownership. Busby:Constellation Pharmaceuticals: Employment, Equity Ownership. Hatton:Constellation Pharmaceuticals: Employment, Equity Ownership. Bryant:Constellation Pharmaceuticals: Employment, Equity Ownership. Trojer:Constellation Pharmaceuticals: Employment, Equity Ownership.

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Combination Treatment With Trabectedin and Irinotecan or Topotecan Has Synergistic Effects Against Ovarian Clear Cell Carcinoma Cells

International Journal of Gynecological Cancer ◽

10.1097/igc.0000000000000143 ◽

2014 ◽

Vol 24 (5) ◽

pp. 829-837 ◽

Cited By ~ 4

Author(s):

Mahiru Kawano ◽

Seiji Mabuchi ◽

Toshiko Kishimoto ◽

Takeshi Hisamatsu ◽

Yuri Matsumoto ◽

...

Keyword(s):

Cell Carcinoma ◽

Cell Lines ◽

Combination Treatment ◽

Clear Cell ◽

Clear Cell Carcinoma ◽

Synergistic Effects ◽

Chemotherapeutic Agents ◽

Ovarian Clear Cell Carcinoma ◽

Combination Treatments

ObjectivesThe objective of this study was to investigate the chemotherapeutic agents that produce the strongest synergistic effects when combined with trabectedin against ovarian clear cell carcinoma (CCC), which is regarded as an aggressive chemoresistant histological subtype.MethodsUsing 4 human CCC cell lines (RMG1, RMG2, KOC7C, and HAC2), the cytotoxicities of trabectedin, SN-38, topotecan, doxorubicin, cisplatin, and paclitaxel as single agents were first assessed using the MTS assay. Then, the cytotoxicities of combination treatments involving trabectedin and 1 of the other 4 agents were evaluated by isobologram analysis to examine whether these combinations displayed synergistic, additive, or antagonistic effects. The antitumor activities of the combination treatments were also examined using cisplatin-resistant and paclitaxel-resistant CCC sublines, which were derived from the parental CCC cells by continuously exposing them to cisplatin or paclitaxel. Finally, we determined the effect of everolimus on the antitumor efficacy of trabectedin-based combination chemotherapy.ResultsConcurrent exposure to trabectedin and SN-38 or topotecan resulted in synergistic interactions in all 4 CCC cell lines. Among the tested combinations, trabectedin plus SN-38 was the most effective cytotoxic regimen. The combination of trabectedin plus SN-38 also had strong synergistic effects on both the cisplatin-resistant and paclitaxel-resistant CCC cell lines. Treatment with everolimus significantly enhanced the antitumor activity of trabectedin plus SN-38 or topotecan.ConclusionsCombination treatment with trabectedin and SN-38 displays the greatest cytotoxic effect against ovarian CCC. Our in vitro study provides the rationale for future clinical trials of trabectedin plus irinotecan with or without everolimus in patients with ovarian CCC in both the front-line chemotherapy setting and as a second-line treatment of recurrent CCC that had previously been treated with cisplatin or paclitaxel.

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Novel Combination Therapy Targeting rDNA Transcription and Histone Deacetylation Provides Effective Treatment for Multiple Myeloma, and Synergises in Bortezomib-Resistant MM

Blood ◽

10.1182/blood-2018-99-113765 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1945-1945

Author(s):

Kylee H Maclachlan ◽

Andrew Cuddihy ◽

Nadine Hein ◽

Carleen Cullinane ◽

Simon J. Harrison ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Drug Resistance ◽

Cell Lines ◽

Cellular Response ◽

Single Agent ◽

Resistant Cell ◽

Rdna Transcription

Abstract Background: Multiple myeloma (MM) requires combination drug therapies to delay acquired drug resistance and clinical relapse. We co-developed CX-5461, a highly-selective inhibitor of RNA polymerase I-mediated rDNA transcription(1), currently in phase I trials for relapsed haematological malignancies (Peter Mac). CX-5461 produces a targeted nucleolar DNA damage response (DDR), triggering both a p53-dependent and -independent nucleolar stress response and killing malignant cells while sparing normal cells(2,3). Single-agent CX-5461 provides an impressive survival benefit in mouse models of B-cell lymphoma, acute myeloid leukaemia and now MM(2,4,5). However, drug resistance eventually occurs, confirming the need for combination therapies. Aim: To test the efficacy of CX-5461 in combination with the histone deacetylase inhibitor panobinostat, (prioritised from a boutique high-throughput screen of anti-myeloma agents), with a focus on the setting of resistance to proteasome-inhibitors (PIs). Methods: We assessed the impact of CX-5461 and panobinostat on overall survival in mouse models of MM, then surveyed the effects on cellular response and molecular markers of DDR. We developed bortezomib-resistant cell lines and an in vivo model of bortezomib-resistance to test this combination in the setting of PI-resistance. Results: CX-5461 in combination with panobinostat provides a significant survival advantage in both the transplanted Vk*MYC and the 5T33/KaLwRij models, with minimal bone marrow toxicity. The combination showed increased anti-proliferative effects and cell death in vitro. Interestingly, experiments interrogating the downstream cellular response of this combination suggest that the mechanism(s) driving synergy are complex and cell context-dependent. Cell cycle analysis indicates that both CX-5461- and panobinostat-driven cell cycle effects, i.e. G2/M and G1/S arrest, respectively, are dominant in the combination setting in a cell line-dependent manner, suggesting that context-dependent factors such as p53 may influence the cellular response. Mechanistically, in both p53-wild type and -null cell lines we observe an increase in DDR signalling with single agent CX-5461, with only moderate further increase with the combination. Moreover, CX-5461-mediated MYC downregulation is not universally observed, with the combination promoting further downregulation only in some cell lines. Given the potential for affecting global transcription programs downstream of panobinostat, we are performing transcriptome analyses in the combination setting compared to single agent treatment. We have generated bortezomib-resistant cell lines, sequentially increasing drug exposure to establish populations growing at concentrations above the IC90 of the parental lines. The resistant 5T33 cells retain their resistance to bortezomib in vivo and we have demonstrated that CX-5461 remains effective in this model, significantly increasing survival. We are currently examining the combination of CX-5461 with panobinostat in this model of bortezomib-resistance, which will give critical information guiding patient selection for future clinical trials. Conclusion: The rDNA transcription inhibitor CX-5461 synergises in vitro and in vivo with panobinostat, and CX-5461 retains efficacy in the setting of bortezomib-resistant myeloma. References Drygin et al., Cancer Research 2011 Bywater et al., Cancer Cell 2012 Quin et al, Oncotarget, 2016 Devlin et al., Cancer Discovery 2016 Hein et al., Blood 2017 Disclosures Harrison: Janssen-Cilag: Other: Scientific advisory board.

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