Characterization of Pim Protein Kinases and Evaluation of Small Molecule Inhibitors in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2909-2909
Author(s):  
Ningfei An ◽  
Yeong-Bin Im ◽  
Yingwei Lin ◽  
Cristina Gasparetto ◽  
Luciano J Costa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Mouse Model ◽  
Protein Kinases ◽  
Cell Lines ◽  
Small Molecule ◽  
Kinase Inhibitors ◽  
Cancer Center ◽  
Myeloma Cells ◽  
Pim Kinases

Abstract Abstract 2909 Multiple myeloma (MM) is a plasma cell malignancy and is the second most common hematological neoplasm in the Western World. Despite the discovery of several highly effective chemotherapy agents, MM remains an incurable disease, suggesting the urgent need for better understanding the disease's pathogenesis and for developing therapeutic agents that target novel molecular pathways. Pim (proviral insertion in murine lymphoma) kinases (Pim-1, -2 and -3) are constitutively active, oncogenic serine/threonine protein kinases that promote early transformation and tumor progression in hematological malignancies and in solid tumors including prostate cancer and colon cancer. In the current study, we characterized Pim expression and investigated the therapeutic potential of small molecule Pim kinase inhibitors in the treatment of MM. We found that Pim kinases are highly expressed in several MM cell lines (NCIH929, OPM-1, RPMI8226, U266 and MM1.R). Furthermore, Pim kinases were up-regulated in freshly isolated primary CD138+ myeloma cells (n = 4) when compared to CD138− cells (panel A). The predominant Pim kinase isoform (Pim-1, -2 or -3) varies among MM cell lines and among different patients. Two Pim kinase inhibitors, SMI-4a (selective small molecule Pim-1 and Pim-3 inhibitor) and 10058-F4 (Pim-3 and Pim-1 inhibitor) effectively inhibited myeloma cell growth, including steroid resistant MM1.R myeloma cells, with IC50s in the ∼10 mM range. Both SMI-4a and 10058-F4 induced apoptotic cell death in myeloma cells as measured by Annexin-V staining, caspase-9 activation and PARP cleavage in a dose dependent manner. To further dissect their mechanisms of action, we investigated the effects of SMI-4a and 10058-F4 on several cellular signaling pathways that are critical to the survival, cell cycle progression and proliferation of MM cells. Our data indicated that both SMI-4a and 10058-F4 reduce c-Myc protein expression levels and down-regulate the antiapoptotic Mcl-1 protein in MM cells (panel B). Our data also showed that MM cells treated with SMI-4a or 10058-F4 significantly reduced mTOR signaling as indicatd by decreased phosphorylation of 4E-BP1 and Cp70 S6 kinase α, the two common mTOR substrates. However, SMI-4a and 10058-F4 had no effect on the cell cycle regulation protein (p27Kip1) or heat shock protein 90. In summary, our data demonstrate that Pim inhibitors affect the survival and proliferation of myeloma cells and represent a potentially effective, novel strategy for the treatment of MM. We are currently performing in vivo studies to evaluate the therapeutic effects of Pim inhibitor in MM animal models (i.e, XPB-1s transgenic mouse model and NOD/SCID xenograft mouse model). This work was supported by MUSC Hollings Cancer Center Startup Fund, Hollings Cancer Center ACS IRG, and ASCO Conquer Cancer Foundation Career Development Award Disclosures: No relevant conflicts of interest to declare.

CHIR258, a Novel Multi-Targeted Tyrosine Kinase Inhibitor, for the Treatment of t(4;14) Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 641-641 ◽  
Author(s):  
Suzanne Trudel ◽  
Zhi Hua Li ◽  
Ellen Wei ◽  
Marion Wiesmann ◽  
Katherine Rendahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Myeloma Cells

Abstract The t(4;14) translocation that occurs uniquely in a subset (15%) of multiple myeloma (MM) patients results in the ectopic expression of the receptor tyrosine kinase, Fibroblast Growth Factor Receptor3 (FGFR3). Wild-type FGFR3 induces proliferative signals in myeloma cells and appears to be weakly transforming in a hematopoeitic mouse model. The subsequent acquisition of FGFR3 activating mutations in some MM is associated with disease progression and is strongly transforming in several experimental models. The clinical impact of t(4;14) translocations has been demonstrated in several retrospective studies each reporting a marked reduction in overall survival. We have previously shown that inhibition of activated FGFR3 causes morphologic differentiation followed by apoptosis of FGFR3 expressing MM cell lines, validating activated FGFR3 as a therapeutic target in t(4;14) MM and encouraging the clinical development of FGFR3 inhibitors for the treatment of these poor-prognosis patients. CHIR258 is a small molecule kinase inhibitor that targets Class III–V RTKs and inhibits FGFR3 with an IC50 of 5 nM in an in vitro kinase assay. Potent anti-tumor and anti-angiogenic activity has been demonstrated in vitro and in vivo. We employed the IL-6 dependent cell line, B9 that has been engineered to express wild-type FGFR3 or active mutants of FGFR3 (Y373C, K650E, G384D and 807C), to screen CHIR258 for activity against FGFR3. CHIR258 differentially inhibited FGF-mediated growth of B9 expressing wild-type and mutant receptors found in MM, with an IC50 of 25 nM and 80 nM respectively as determined by MTT proliferation assay. Growth of these cells could be rescued by IL-6 demonstrating selectivity of CHIR258 for FGFR3. We then confirmed the activity of CHIR258 against FGFR3 expressing myeloma cells. CHIR258 inhibited the viability of FGFR3 expressing KMS11 (Y373C), KMS18 (G384D) and OPM-2 (K650E) cell lines with an IC50 of 100 nM, 250 nM and 80 nM, respectively. Importantly, inhibition with CHIR258 was still observed in the presence of IL-6, a potent growth factors for MM cells. U266 cells, which lack FGFR3 expression, displayed minimal growth inhibition demonstrating that at effective concentrations, CHIR258 exhibits minimal nonspecific cytotoxicity on MM cells. Further characterization of this finding demonstrated that inhibition of cell growth corresponded to G0/G1 cell cycle arrest and dose-dependent inhibition of downstream ERK phosphorylation. In responsive cell lines, CHIR258 induced apoptosis via caspase 3. In vitro combination analysis of CHIR258 and dexamethasone applied simultaneously to KMS11 cells indicated a synergistic interaction. In vivo studies demonstrated that CHIR258 induced tumor regression and inhibited growth of FGFR3 tumors in a plasmacytoma xenograft mouse model. Finally, CHIR258 produced cytotoxic responses in 4/5 primary myeloma samples derived from patients harboring a t(4;14) translocation. These data indicate that the small molecule inhibitor, CHIR258 potently inhibits FGFR3 and has activity against human MM cells setting the stage for a Phase I clinical trial of this compound in t(4;14) myeloma.

Initial Evaluation of Novel Dual PIM/PI3K and Triple PIM/PI3K/mTOR Inhibitors in Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5713-5713 ◽  
Author(s):  
Mairead Reidy ◽  
Marianne vanDijk ◽  
Niamh Keane ◽  
Michael O'Neill ◽  
Michael E O'Dwyer
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Mouse Model ◽  
Cell Viability ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Annexin V ◽  
Mtor Inhibitors ◽  
Pim Kinases

Abstract Background: Despite advances in treatment, Multiple Myeloma (MM) remains incurable. The PI3K/AKT pathway is activated in MM cells in > 50% of cases due to factors such as bone marrow (BM) microenvironmental signaling and hyperactivation following treatment with proteasome inhibitors (PI). Multiple small-molecule inhibitors have been developed to target PI3K/AKT or mTOR kinases, but the efficacy of these drugs is likely to be compromised by the stimulation of compensatory signaling pathways. The redundancy of signaling pathways provides back-up mechanisms allowing escape from targeted inhibition. One such compensatory pathway is that driven by PIM kinases, which produce parallel oncogenic signals to AKT and mTOR and share several downstream molecular targets. As with PI3K/AKT, the BM microenvironment plays a major role in PIM activation and other factors increasing PIM levels include hypoxia and PI treatment. PIM1 and particularly PIM2 are known to be highly expressed in MM and play important roles in regulating MYC-driven transcription, apoptosis, cytokine signaling, cell proliferation and protein translation. Combinations of separate PI3K and PIM inhibitors have shown evidence of synergy in MM cell lines and animal models and a PIM kinase inhibitor has recently shown activity in relapsed/refractory MM. Given this background we wished to evaluate the activity of a novel family of kinase inhibitors capable of inhibiting not only PIM kinases but also PI3K/AKT (dual inhibitors) and PI3K/AKT/mTOR (triple inhibitors). Methods: We evaluated the in-vitro activities of a single pan-PIM (pPIMi), dual PIM/PI3K (IBL-202) and triple PIM/PI3K/mTOR (IBL-301) inhibitor in MM cell lines: MM1.S, NCI-H929, RPMI8226 and KMS11, which is known to be PIM2 dependent, alongside the pan-PI3K inhibitor GDC-0941 and the pan-PIM inhibitor AZD1208. IBL-202 and IBL-301 are optimized lead compounds and are low nanomolar pan-PIM/PI3K and pan-PIM/PI3K/mTOR inhibitors respectively. These dual and triple inhibitors show excellent kinase selectivity profile against a panel of 456 kinases. Cell viability was assessed using the Cell-Titre Glo assay and apoptosis determined by Annexin-V/PI staining. Co-culture experiments were performed with HS-5 stromal cells. Combination treatment was performed with bortezomib and IBL-202 to assess synergy. Results and discussion: IBL-202 and IBL-301 were significantly more potent than pPIMi in all MM cell lines tested (figure 1). IBL-202 and IBL-301 caused a loss in cell viability 50% and 70%, respectively, greater than pPIMi alone. IBL-202 and IBL-301 induced 50-80% and 80-100% cell death, respectively .v. 10% for pPIMi after 48 hrs, p<0.001. The Pim2 dependent MM cell line KMS11 showed a loss in cell viability following treatment with IBL-202 and IBL-301 up to three times greater than either of the PIM kinase inhibitors or GDC-0941. IBL-202 treatment caused a 90% reduction in cell viability at a dose of 5µM and IBL-301 was equally effective at a concentration of just 1µM. GDC-0941(5µM) caused a loss of approximately 30% in cell viability whereas cells remained entirely resistant to pPIMi and AZD1208 at concentrations up to 10µM (p< 0.001). IBL-202 in combination with bortezomib was synergistic in MM cell lines (CI<1). While co-culture with HS-5 cells protected MM cell lines against bortezomib-induced cell death, it promoted the apoptotic effect of both IBL-202 and IBL-301 with an increase in Annexin V positive cells from 15% to 40%. This suggests that micro-environmental stimulation could potentially induce synthetic lethality in the presence of these inhibitors. We observed strong induction of PIM2 in MM1.S cells following co-culture. Mechanistically, cells respond to dual and triple inhibitors with cell cycle arrest, marked apoptosis and strong down-regulation of biomarkers. The dual and triple inhibitors are optimized with respect to their in vitro ADME properties and have excellent oral bioavailability. In-vivo IBL-301 has been well tolerated, with no signs of toxicity even 20 times above the efficacious dose in a transgenic (KRASV12NSCLC) mouse model. Testing of IBL-202 in a relevant MM mouse model is planned in the near future. Conclusions: IBL-201 and IBL-301 show promising activity in MM cellular models with increased potency compared to inhibitors targeting PIM or PI3K alone and warrant further evaluation in this disease. Figure 1. Figure 1. Disclosures O'Neill: Inflection Biosciences: Employment, Equity Ownership. O'Dwyer:Inflection Biosciences: Membership on an entity's Board of Directors or advisory committees.

Dual Antitumoral and Bone Antiresorptive Effect Of The Pan-Pim Kinase Inhibitor, LGH447, In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4435-4435
Author(s):  
Teresa Paíno ◽  
Antonio Garcia-Gomez ◽  
Lorena González-Méndez ◽  
Laura San-Segundo ◽  
Montserrat Martín-Sánchez ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Myeloma Cells ◽  
Pim Kinases ◽  
Castilla Y Leon ◽  
Ic50 Values ◽  
Rpmi 8226 ◽  
Resorptive Activity

Introduction Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells in the bone marrow (BM) and is closely associated with osteolytic lesions, in part due to an increase in the bone-resorptive activity and number of osteoclasts (OCs). The activation of survival pathways in myeloma cells could be the cause of treatment failure rendering the disease incurable. Pim kinases are a family of survival serine/threonine kinases composed of three members (Pim1, Pim2 and Pim3) that are overexpressed in MM cells and may have a role in MM pathogenesis. However, little is known about the role of Pim kinases in OCs and its involvement in myeloma bone disease. Here, we have evaluated the preclinical activity of a new pan-Pim kinase inhibitor, LGH447, on MM cells and OCs. Cell lines, primary samples, material and methods LGH447 was provided by Novartis Pharmaceuticals. The human MM cell lines MM1S, MM1R, RPMI-8226 (or RPMI-8226-luc), RPMI-LR5, MM144, NCI-H929, OPM-2, U266, U266-Dox4 and U266-LR7 were employed. PBMCs from healthy volunteers were used to generate OCs, whereas primary mesenchymal stromal cells (MSCs) were obtained from bone marrow aspirates of MM patients. Cell viability was studied using MTT colorimetric assay or bioluminescence. Apoptosis was measured by annexin-V staining. For cell cycle analysis, propidium iodide staining was used. OC formation was assessed by enumeration of multinucleated (≥3) TRAP-positive cells and OC resorption was assessed on calcium-coated slides. Immunoblotting, quantitative PCR and immunofluorescence were used to further investigate the mechanism of action of LGH447. Results All MM cell lines expressed the three isoforms of Pim kinases with higher levels of Pim2. The dose-response curves to LGH447 after a 48 hour treatment revealed two groups of MM cell lines with regard to sensitivity to this drug: high sensitive, with IC50 values ranging from 0.2 to 3.3 µM (MM1S, MM1R, RPMI-8226, MM144, U266 and NCI-H929); and low sensitive, with IC50 values >7 µM (OPM-2, RPMI-LR5, U266-Dox4 and U266-LR7). Our results indicated that LGH447 promoted apoptosis in myeloma cells as shown by the increase in annexin-V positive cells and by the cleavage of initiator (caspases 8 and 9) and effector caspases (caspases 3 and 7) and of PARP. LGH447 also blocked the cell cycle in MM cells as demonstrated by the increase in G0-G1 and the decrease in S-G2-M phases. Importantly, LGH447 was also able to overcome the growth advantage conferred to RPMI-8226-luc cells by co-culture with MSCs or OCs. Regarding the mechanisms involved in these effects, LGH447 inhibited the mTOR pathway, demonstrated by a decreased phosphorylation of the downstream mTOR effectors, 4EBP1 and S6 in residues Thr37/46 and Ser235/236, respectively. Interestingly, LGH447 also inhibited OC formation and resorption activity. LGH447 treatment of human pre-OCs diminished the expression of key molecules involved in OC differentiation (p-Erk1/2 and NFATc1) and function [CAII (carbonic anhidrase II), CLCN7 (chloride channel 7), ATP6V1A (vacuolar-H+-ATPase catalytic subunit A1) and MMP9 (matrix metalloproteinase 9)] and also disrupted the F-actin ring necessary for OC effective resorption. Conclusion Overall, our results demonstrate that both MM cells and OCs are targets of the pan-Pim kinase inhibitor, LGH447. Therefore, the inhibition of Pim kinases could potentially provide a dual benefit in myeloma patients as a consequence of cytotoxic effects exerted on MM cells and an anti-resorptive activity on bone. This work was supported by funding from the Fundación Española de Hematología y Hemoterapia (AG-G), Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, the RTICC-Hematology Group (RD12/0036/0058), Spanish FIS (PI12/02591) and the Junta de Castilla y León, Gerencia Regional de Salud (GRS 862/A/13). Disclosures: Off Label Use: LGH447 is a pan-Pim kinase inhibitor (Novartis Pharmaceuticals). It has been used for pre-clinical studies in multiple myeloma.

Valproic Acid Inihibts Histone Deacetylases and Proliferation and Induces Cell Cycle Arrest and Apoptosis in Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5165-5165
Author(s):  
Martin Kaiser ◽  
Ulrike Heider ◽  
Ivana Zavrski ◽  
Jan Sterz ◽  
Kurt Possinger ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Valproic Acid ◽  
Cell Lines ◽  
Myeloma Cell ◽  
G1 Phase ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Cycle Arrest ◽  
Dose Dependent

Abstract Multiple myeloma remains an incurable disease in the majority of the patients and novel treatment strategies are urgently needed. A new class of drugs, the histone deacetylase (HDAC) inhibitors take influence in epigenetic modifications and have antiproliferative effects in some malignancies. Valproic acid (VPA) is an anticonvulsant drug and was recently shown to inhibit HDACs and suppress tumor growth. The drug is currently being evaluated in clinical studies in acute myeloid leukemia. Its effects on myeloma cells are unknown. The aim of this study was to evaluate the effects of VPA on proliferation, apoptosis and HDAC inhibition in multiple myeloma cell lines as well as in sorted human bone marrow multiple myeloma cells. Myeloma cell lines, OPM-2, NCI-H929, LP-1, and freshly isolated multiple myeloma cells from bone marrow aspirates were exposed to different concentrations of VPA for 4 to 72 hours. Cell proliferation, cell cycle distribution and apoptosis were assayed in reaction to the treatment. Proliferation decreased noticeably and apoptosis was induced in a dose-dependent manner in multiple myeloma cell lines as well as in freshly sorted primary myeloma cells. After 48 hours of incubation with VPA at 1 mM, approximately 46%, 52% and 25% of OPM-2, NCI-H929 and LP-1 cell lines had undergone specific apoptosis, respectively. Freshly sorted primary bone marrow myeloma cells from patients showed also specific apoptosis. In cell cycle analysis by flow cytometry, the population of cells in the G0/G1 phase increased, whereas cells in the S phase decreased in a time and dose dependent manner. Incubation of the cell line OPM-2, for example, with 1 mM VPA for 48 hours decreased the proportion of cells in the S phase from 39 % to 6 % of the total cell count and increased cells in the G0/G1 phase from 49 % to 85 %. Acetylation of histones and expression of cyclin D1 and the cell cycle regulators p21 and p27 were studied by western blot. Histone acetylation and p21 concentrations increased after VPA treatment whereas levels of p27 remained constant. A decrease in cyclin D1 concentrations was observed. Subapoptotic doses of VPA significantly decreased the production of VEGF in OPM-2 cell line. These data show that treatment with valproic acid effectively inhibits histone deacetylase activity, leading to the accumulation of acetylated histones in multiple myeloma cells. Parallel upregulation of cell cycle inhibitors like p21WAF1 was observed, together with a reduction of cyclin D1 levels. Myeloma cell proliferation was inhibited in a time and dose dependent manner and cell cycle arrest in the G0/G1 phase was induced by VPA treatment. VPA potently induced apoptosis in all human myeloma cell lines as well as in sorted primary multiple myeloma cells in a dose and time dependent manner. These results show for the first time that VPA acts as an HDAC inhibitor in multiple myeloma cells, induces G1 cell cycle arrest, potently inhibits tumor growth and markedly induces apoptosis. In addition to its direct antitumor effect, valproic acid may exert an antiangiogenic effect by reducing VEGF production in myeloma cells. These data provide the framework for clinical studies with valproic acid in multiple myeloma.

Myeloma Cell Proliferation Is Inhibitied by the Activation of Adenosine Monophosphate Activated Protein Kinase (AMPK).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5045-5045
Author(s):  
Philipp Baumann ◽  
Sonja Mandl-Weber ◽  
Bertold Emmerich ◽  
Christian Straka ◽  
Daniel Franke ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Adenosine Kinase ◽  
Intracellular Signalling ◽  
Phase Cell ◽  
Myeloma Cells ◽  
Multiple Myeloma Cell

Abstract In multiple myeloma (MM), a network of cytokines in the bone marrow microenvironment promotes myeloma cell proliferation. Consequent inhibition of intracellular signalling in the myeloma cells seems to be a promising strategy to encounter disease progression. The multiple myeloma cell lines U266, OPM-2, RPMI-8226 and NCI-H929 were incubated with the AMPK activators AICAr and D942. Basal and cytokine stimulated proliferation rates of myeloma cells were measured by the WST-1 assay. Alterations of the cell cycle were determined by flow cytometry after staining with propidium iodide. Intracellular signalling was shown by western blotting. The AMPK activators 5-aminoimidazole-4-carboxamide (AICAr) and D942 induced inhibition of proliferation in multiple myeloma cell lines. AICAr also induced a S-phase cell cycle arrest in all four tested cell lines and led to phosphorylation and herewith activation of AMPK. Furthermore, the inhibition of a nucleoside transporter by nitrobenzyl-thio-9-β-D-ribofuranosylpurine (NBTI), inhibition of the adenosine kinase by iodotubericidine and inhibition of AMPK by AMPKI Compound C reversed AICAr effects, indicating that the cellular effects of AICAr were mediated by AMPK. Activation of AMPK inhibited basal extracellular-signal regulated kinase (ERK), mTOR and P70S6 kinase (P70S6K) signalling and blocked cytokine induced increase of proliferation, which again was due to inhibition of ERK and P70S6K signalling. Troglitazone, a representative of a group of anti-diabetic drugs, similarly inhibited myeloma cell proliferation, activated AMPK and decreased ERK and P70S6K signalling. We demonstrate for the first time that myeloma cell proliferation is controlled by AMPK activity. Consequently, targeting this pathway by inhibitors like glitazones provides a novel strategy in myeloma therapy.

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

Blood ◽  
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.

The Dual PIM/PI3-K Inhibitor Ibl-202 Overcomes Microenvironmental Mediated Resistance in Multiple Myeloma and Prevents PIM1 Induced CXCR4 Upregulation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5350-5350
Author(s):  
Mairead Reidy ◽  
Marianne VanDijk ◽  
Michael O'Neill ◽  
Michael O'Dwyer
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Surface Expression ◽  
Down Regulation ◽  
Pim Kinases ◽  
Ic50 Values ◽  
And Migration

Abstract Background: The interaction of multiple myeloma (MM) cells with bone marrow (BM) cells along with factors in the BM milieu such as chemokines and cytokines play a crucial role in both progression of MM and drug resistance. Activation of the PI3-K/Akt survival pathway is a characteristic of both human MM cell lines and patient samples. This activation can be linked to BM microenvironmental signalling and use of proteasome inhibitors in treatment, suggesting this as a crucial point of therapeutic intervention to abrogate growth and survival signals in MM. However, the efficacy of such therapeutics has been modest and is likely to be compromised by the stimulation of compensatory signalling pathways, such as the PIM kinases, which like the PI3-K/Akt pathway are also induced by BM microenvironmental influences and share similar downstream targets. These proto-oncogenic kinases are constitutively active and play an important role in proliferation and survival in MM. The influence of these kinases on homing and migration has been observed in other malignancies, this has yet to be reported in MM. Here we report the effects of a dual inhibitor of PIM/PI3-K, IBL-202, and provide novel insights into effects on cell survival, signaling and migration. Methods: We investigated the effect of IBL-202 against a panel of MM cell lines (MM.IS, NCI-H929s, KMS11 and RPMI-8226) and primary MM patient samples. The in vitro efficacy of IBL-202 was compared to that of single pan-PIM inhibitors pPIMi and AZD1208 and also the pan-PI3-K inhibitor GDC-0941. Apoptosis was measured with AnnexinV staining and cell cycle analysed with Edu/DAPI staining. To mimic BM microenvironmental conditions MM cells were cultured under hypoxic conditions (1% O2) and in co-culture with the human stromal cell line HS5. Surface expression of CXCR4 was assessed in MM cell lines by flow cytometry. PIM kinases, pCXCR4 and downstream targets of PIM/PI3-K were examined by western blot. Transwell migration assays were carried out in the presence of 50ng SDF-1α for 4h @ 37o C. Results: Simultaneous inhibition of PIM and PI3-K using IBL-202 in vitro was significantly more potent at inducing apoptosis than GDC-0941, pPIMi or AZD1208 in all MM cell lines tested. IC50 values were under 1μM for IBL-202 at 48h whilst in comparison the pan PIM inhibitors pPIMi and AZD1208 scored IC50 values between 5 and 10μM. The IC50 for GDC-0941 was on average 5μM (Figure 1). At the molecular level there was a notable decrease in phosphorylation of known PIM/PI3-K targets Akt (Ser473), Bad (Ser112) and the translational targets S6 (Ser235/236) and 4EBP1 (Thr37/46). The levels of total proteins were unchanged. Treatment with increasing doses of IBL-202 led to a marked reduction in cells in S phase of the cell cycle. These changes were paralled by down regulation of the cell cycle promoting proteins cyclin D1 and c-myc. IBL-202 was also effective in inducing apoptosis in primary MM patient samples (n=4) after just 24h as assessed by Annexin-V staining (Figure 2). To explore the role of the BM microenvironment we co-cultured MM cell lines with HS5s. This led to strong induction of PIM2 in MM cells. While MM cells in this setting were protected from Bortezomib-induced cell death, the apoptotic effect of IBL-202 was enhanced. In a further effort to mimic the tumour microenvironment we cultured MM cell lines in hypoxia. This may be of particular relevance as Pim-1 has been reported to be a pivotal regulator involved in hypoxia-induced chemoresistance. MM cells were further sensitised to IBL-202 in hypoxia. In addition, hypoxia increased the surface expression of CXCR4, a chemokine receptor critical for homing of MM cells to the bone marrow, with a concomitant increase in PIM1. Treatment of MM cell lines with IBL-202 reduced the level of PIM1 and CXCR4 Ser339 phosphorylation, along with down regulation of CXCR4 surface expression resulting in reduced migration of MM cells along an SDF-1 gradient. Conclusion: Together these data provide direct evidence of the potency of IBL-202 in MM in conditions that mimic the BM microenvironment. Moreover, they indicate a potential role for PIM kinases in facilitating dissemination and invasiveness of MM by CXCR4 and provide an added rationale for targeting PIM kinases in MM. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures O'Neill: Inflection Biosciences: Employment.

A Novel Orally Active Small Molecule Potently Induces G1 Arrest in Primary Myeloma Cells and Prevents Tumor Growth by Specific Inhibition of Cdk4/6.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 369-369 ◽  
Author(s):  
Linda B. Baughn ◽  
Maurizio Di Liberto ◽  
Kaida Wu ◽  
Peter Toogood ◽  
Tracey Louie ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Tumor Growth ◽  
Small Molecule ◽  
Critical Role ◽  
Cellular Transformation ◽  
G1 Arrest ◽  
Specific Inhibition ◽  
Myeloma Cells ◽  
Orally Active

Abstract Cell cycle deregulation is central to the initiation and fatality of multiple myeloma, the second most common hematopoietic cancer, although impaired apoptosis plays a critical role in the accumulation of myeloma cells in the bone marrow (BM). Inhibition of Cdk4 and Cdk6 by the Cdk inhibitor, p18(INK4c), is required for the generation of normal, functional plasma cells1. The mechanism for intermittent, unrestrained proliferation of myeloma cells is unknown, but mutually exclusive activation of Cdk4-cyclin D1 or Cdk6-cyclin D2 precedes proliferation of BM myeloma cells in vivo2. These data identify Cdk4 and Cdk6 as key determinants in the loss of cell cycle control in myeloma and suggest that Cdk4/6 may be effective targets for therapeutic intervention. Here we show that by specific inhibition of Cdk4/6, the orally active small molecule PD 0332991 potently induces G1 arrest in primary BM myeloma cells ex vivo, and prevents tumor growth in disseminated human myeloma xenografts. PD 0332991 inhibits Cdk4/6 proportional to the cycling status of the cells independent of cellular transformation, and acts in concert with the physiologic Cdk4/6 inhibitor p18(INK4c). Inhibition of Cdk4/6 by PD 0332991 is not accompanied by induction of apoptosis. However, when used in combination with a second agent such as dexamethasone, PD 0332991 markedly enhances the killing of myeloma cells by dexamethasone. PD 0332991, therefore, represents the first promising and specific inhibitor for therapeutic targeting of Cdk4/6 in multiple myeloma and possibly other B cell cancers.

Trolox Improves the Sensitivity of Multiple Myeloma to Arsenic Trioxide in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4920-4920
Author(s):  
Michael Sebag ◽  
Xian-Fang Huang ◽  
Nicolas Garnier ◽  
Wilson H. Miller ◽  
Koren Mann
Keyword(s):  
Oxidative Stress ◽  
Multiple Myeloma ◽  
Cell Growth ◽  
Mouse Model ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Malignant Cells ◽  
Myeloma Cells

Abstract Abstract 4920 Arsenic trioxide (ATO) induces apoptosis and promotes differentiation of acute promyelocytic leukemia (APL) cells, but has less activity in other types of cancers. One factor that may impede ATO success outside of APL is its toxicity profile, which limits in vivo concentrations and therefore, therapeutic benefit. We have reported that trolox, an analogue of alpha tocopherol, can augment ATO sensitivity in a variety of malignant cells, while protecting non-malignant cells from ATO toxicity. In this current study, we have focused on Multiple Myeloma (MM), a plasma cell malignancy that often shows resistance to apoptosis, drug inhibition and remains incurable despite tremendous recent advances. Although ATO has activity against MM cells in vitro, clinical trials of ATO, given as a solo agent, in MM have shown limited promise. To see if the addition of trolox could augment ATO toxicity, a panel of human myeloma cell lines (HMCLs, n=9) representing the genetic diversity seen in this disease, were treated with increasing concentration of ATO with and without 100uM trolox. Cell growth was assessed by MTT viability assays and virtually all cell lines were sensitive to varying doses of ATO. Four cell lines (U266, KMS11, MM1R, MM1S) showed profound inhibition of cell growth with very low concentrations of ATO (<1uM). Trolox (100uM) alone had no effect on cell growth, but in concert with ATO further decreased cell growth by up to 50% as compared to the same dose of ATO alone in virtually all cell lines. To further elucidate the mechanism of growth inhibition, annexin V assays were performed by flow cytometry to measure apoptosis. In all cell lines (n=9), a clear increase in the apoptotic fraction was noted when trolox was added to varying doses of arsenic. To test whether oxidative stress plays a role in ATO-mediated apoptosis of myeloma cells, we looked at the induction of a stress response protein (HO-1), a marker of oxidative stress induced by ATO. Western blot analysis revealed that in all myeloma cells tested, HO-1 was dramatically and quickly induced by ATO and further induced by the addition of trolox, indicating a pro-oxidant activity of trolox in the malignant cells. While the mechanism of trolox enhancement of ATO function remains largely unknown, intracellular concentrations of ATO in MM cells, as measured by inductively coupled plasma mass spectrometry, suggest that trolox does not work by augmenting ATO import or intracellular accumulation. To test the efficacy of ATO with trolox in vivo, we used a novel transgenic mouse model of MM that has been shown to faithfully mimic the human disease and its response to treatment (Chesi et al, Cancer Cell 2008 Feb;13(2):167-80). We first treated MM afflicted mice with a low dose of ATO (5.0mg/kg) and Trolox (50mg/kg) to assess for toxicity and tolerability. This dose was well tolerated in all mice when given for 10 days with no obvious toxic effects. Serum protein electrophoresis performed at the end of the 10 day treatment period revealed that even at this low starting dose, one of three mice showed a 30% reduction in its paraprotein peak, while the others remained stable. Further studies with higher ATO concentrations in the same mouse model are underway. In conclusion, these data support the role of ATO plus Trolox, as a promising anti-myeloma therapy. Disclosures No relevant conflicts of interest to declare.

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