scholarly journals SYK-Inhibitor Bay 61-3606 Induces Cell Cycle Arrest and Apoptosis in Multiple Myeloma Cells Independent of SYK Inhibitory Effects but Via Degradation of IKZF1/3

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4477-4477
Author(s):  
Shirong Li ◽  
Jing Fu ◽  
Jing Wu ◽  
Markus Y Mapara ◽  
Suzanne Lentzsch
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Western Blot ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Western Blot Assay ◽  
Myeloma Cells ◽  
Cycle Arrest ◽  
Syk Inhibitor ◽  
Rpmi 8226

Abstract Introduction: Bay 61-3606 is a cell-permeable imidazopyrimidine compound that acts as a potent, ATP-competitive, reversible, and highly selective inhibitor of Syk tyrosine kinase activity with no inhibitory effect against Btk, Fyn, Itk, Lyn, and Src. BAY 61-3606 has been also shown to inhibit Syk-mediated cellular functions such as glucose-tyrosine phosphorylation of I κ B α and p65 nuclear translocation. It further exhibits a good oral bioavailability and in vivo efficacy in rat models. Recently, Bay 61-3606 was found to inhibit cell proliferation and SDF-1a-induced migration of MM cells (1). Based on these promising preliminary data we further investigated the potential of Bay 61-3606 as new anti-MM agent. Methods and Results: Bay 61-3606, at concentrations as low as 10 nM, induced significant (p<0.01) inhibition of cell growth in MM cells (MM.1S, H929 and RPMI-8266) as shown by WST-1 cell proliferation assay. The inhibition of proliferation was accompanied by increased cell cycle arrest at G0/G1 (from 60% to 75%). More importantly, Bay 61-3606 dose dependently induced MM cells apoptosis suggesting that Bay 61-3606 has potent anti-MM properties. Since Bay 61-3606 was originally reported as a SYK inhibitor, we analyzed whether the effects of Bay 61-3606 were due to SYK inhibition. For this purpose, we first checked the expression of SYK in all the MM cell lines (MM.1S, H929, U266, OPM2 and RPMI-8226). And to our surprise, although all these cells were sensitive to Bay 61-3606 treatment, several of these cell lines (H929, U266 and RPMI-8226) had no detectable SYK expression by Western Blot assay. This suggests that the anti-MM effects of Bay 61-3606 are not mediated by SYK inhibition, which was further confirmed by using another specific SYK inhibitor GS-9973. Further mechanistic studies showed that Bay 61-3606 significantly downregulated protein levels of IKZF1, IKZF3, c-MYC and IRF-4 in IMiD®-sensitive and resistant MM cell lines (MM.1S, H929 and RPMI-8266) by Western Blot assay. In contrast, real-time PCR assay indicated IKZF1/3-mRNA level was not altered, suggesting that Bay 61-3606 regulates IKZF1/3 at post-translational level. The proteinase inhibitor MG132 and cullin-dependent ubiquitin ligase inhibitor MLN4924 blocked the downregulation IKZF1/3 by Bay 61-3606 confirming that Bay 61-3606 induced IKZF1/3 protein degradation. Consistent with its proapoptotic effects, Bay 61-3606 downregulated MDM2 phosphorylation, upregulated P53 level and induced PARP cleavage as confirmed by Western Blot assy. Conclusion: In summary, our results demonstrate that Bay 61-3606 has anti-MM effects by inducing cell cycle arrest and apoptosis, and this effect is independent of SYK inhibition. Similar to IMiD® compounds, Bay 61-3606 induces IKZF1 and IKZF3 protein degradation in MM cells. More importantly, Bay 61-3606 has effects on both IMiD®-sensitive and resistant myeloma cells and may represent a novel potent anti-MM agent. References: (1). Koerber RM, et al. Exp Hematol Oncol. 2015. Disclosures Lentzsch: BMS: Consultancy; Foundation One: Consultancy; Celgene: Consultancy, Honoraria.

scholarly journals Curcumin Reverses NNMT-Induced 5-Fluorouracil Resistance via Increasing ROS and Cell Cycle Arrest in Colorectal Cancer Cells

Biomolecules ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1295
Author(s):  
Guoli Li ◽  
Sining Fang ◽  
Xiao Shao ◽  
Yejia Li ◽  
Qingchao Tong ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Induced Resistance ◽  
Phase Cell ◽  
Ros Generation ◽  
Antitumor Effects ◽  
Cycle Arrest

Nicotinamide N-methyltransferase (NNMT) plays multiple roles in improving the aggressiveness of colorectal cancer (CRC) and enhancing resistance to 5-Fluorouracil (5-FU), making it an attractive therapeutic target. Curcumin (Cur) is a promising natural compound, exhibiting multiple antitumor effects and potentiating the effect of 5-FU. The aim of the present study is to explore the effect of Cur on attenuating NNMT-induced resistance to 5-FU in CRC. A panel of CRC cell lines with different NNMT expressions are used to characterize the effect of Cur. Herein, it is observed that Cur can depress the expression of NNMT and p-STAT3 in CRC cells. Furthermore, Cur can induce inhibition of cell proliferation, G2/M phase cell cycle arrest, and reactive oxygen species (ROS) generation, especially in high-NNMT-expression CRC cell lines. Cur can also re-sensitize high-NNMT-expression CRC cells to 5-FU both in vitro and in vivo. In summary, it is proposed that Cur can reverse NNMT-induced cell proliferation and 5-FU resistance through ROS generation and cell cycle arrest. Given that Cur has long been used, we suppose that Cur is a promising anticancer drug candidate with minimal side effects for human CRC therapy and can attenuate NNMT-induced resistance to 5-FU.

scholarly journals Diallyl thiosulfinate enhanced the anti-cancer activity of dexamethasone in the side population cells of multiple myeloma by promoting miR-127-3p and deactivating the PI3K/AKT signaling pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wenfeng He ◽  
Yonghui Fu ◽  
Yongliang Zheng ◽  
Xiaoping Wang ◽  
Bin Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Cycle Arrest ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Colony Formation ◽  
Side Population ◽  
Mtor Signaling Pathway ◽  
Cycle Arrest

Abstract Background Side population (SP) cells, which have similar features to those of cancer stem cells, show resistance to dexamethasone (Dex) treatment. Thus, new drugs that can be used in combination with Dex to reduce the population of SP cells in multiple myeloma (MM) are required. Diallyl thiosulfinate (DATS, allicin), a natural organosulfur compound derived from garlic, has been shown to inhibit the proliferation of SP cells in MM cell lines. Therefore, we investigated the effect of a combination of DATS and Dex (DAT + Dex) on MM SP cells. Methods SP cells were sorted from MM RPMI-8226 and NCI-H929 cell lines using Hoechst 33342-labeled fluorescence-activated cell sorting. The growth of SP cells was evaluated using the cell counting kit-8 assay. Cell cycle and apoptosis assays were conducted using a BD Calibur flow cytometer. miRNA expression was measured using quantitative reverse transcription-polymerase chain reaction. Phosphoinositide 3-kinase (PI3K), phosphorylated AKT (p-AKT), AKT, p-mechanistic target of rapamycin (mTOR), and mTOR levels were measured using western blot analysis. Results Our results showed that the combination of DATS+Dex inhibited sphere formation, colony formation, and proliferation of MM SP cells by inducing apoptosis and cell cycle arrest in the G1/S phase. In addition, the combination of DATS+Dex promoted miR-127-3p expression and inhibited PI3K, p-AKT, and p-mTOR expression in SP cells. Knockdown of miR-127-3p expression weakened the effect of DATS+Dex on cell proliferation, colony formation, apoptosis, and cell cycle of MM SP cells. Additionally, knockdown of miR-127-3p activated the PI3K/AKT/mTOR signaling pathway in MM SP cells cotreated with DATS+Dex. Conclusion We demonstrated that cotreatment with DATS+Dex reduced cell proliferation, promoted apoptosis, and caused cell cycle arrest of MM SP cells by promoting miR-127-3p expression and deactivating the PI3K/AKT/mTOR signaling pathway.

scholarly journals Cell cycle arrest following pan-RAS inhibition can occur through upregulation of p27 in both KRAS-dependent and KRAS-independent colon cancer cell lines

2020 ◽  
Author(s):  
Caleb K. Stubbs ◽  
Marco Biancucci ◽  
Vania Vidimar ◽  
Karla J. F. Satchell
Keyword(s):  
Cell Cycle ◽  
Colon Cancer ◽  
Cell Proliferation ◽  
Growth Inhibition ◽  
Cell Cycle Arrest ◽  
Cancer Cells ◽  
Cell Lines ◽  
Ras Signaling ◽  
Colon Cancer Cells ◽  
Cycle Arrest

ABSTRACTRAS is one of the most frequently mutated oncogenes in cancer with ~30% of all human tumors harboring a mutation in either HRAS, NRAS, or KRAS isoforms. Despite countless efforts for development of small molecule inhibitors for RAS, it remains an elusive target in the clinic. Here we demonstrated that the pan-RAS biological inhibitor RAS/RAP1-specific endopeptidase (RRSP) has proteolytic activity in ‘Ras-less’ mouse embryonic fibroblasts expressing human RAS isoforms (H/N/KRAS) or major oncogenic KRAS mutants (G12C, G12V, G12D, G13D, and Q61R). The cleavage of RAS inhibited phosphorylation of ERK and cell proliferation. To investigate how RAS processing affects colon cancer cells, we tested RRSP against KRAS-dependent (SW620 and GP5d) and KRAS-independent (HCT-116, SW1463, and HT-29) cell lines and found that RRSP inhibited growth. The cleavage of RAS was cytotoxic in some cell lines and induced either irreversible cell cycle arrest or uncharacterized growth inhibition in others. The G1 cell cycle arrest in some colon cancer cells was mediated through rescue of p27 (Kip1) protein expression resulting in reduced phosphorylation of retinoblastoma protein. Together, this work demonstrated that complete ablation of RAS in cells induces growth inhibition, but the mechanism of inhibition can vary in different tumor cell lines. This ability of RAS processing to halt cell proliferation by multiple strategies highlights RRSP both as a potential anti-tumor therapy and as a tool for studying RAS signaling across tumor types.

Imipramine Blue, a Tricyclic Compound and An Inhibitor of NADPH Oxidase Induces G2/M Cell Cycle Arrest Followed by Apoptosis in Multiple Myeloma Cells and in Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5170-5170
Author(s):  
Ebenezer David ◽  
Jonathan L Kaufman ◽  
Jack Arbiser ◽  
Sagar Lonial
Keyword(s):  
Cell Cycle ◽  
Nadph Oxidase ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Myeloma Cell ◽  
M Cell ◽  
Myeloma Cells ◽  
Cycle Arrest ◽  
Pi3 Kinase Pathway ◽  
Kinase Pathway

Abstract Background: Multiple myeloma is an incurable malignancy of plasma cells. While conventional and novel therapies have improved the outcome of patients with myeloma, new therapies and therapeutic targets are needed. NADPH oxidase represents a potential therapeutic target, as NADPH oxidase inhibitors block transduction of signals from cell surface receptors and the PI3 kinase pathway that are dependent on reactive oxygen species (ROS). We tested the effect of the NADPH oxidase inhibitor, Imipramine Blue (IB), on myeloma cell growth and activation of apoptosis in cell lines and in primary human myeloma cells. Methods: IB was synthesized by refluxing imipramine and michlers ketone in the presence of phosphorus oxychloride. MTT assays for myeloma cells (MM.1S, MM.1R, RPMI8226, and U266) were used to evaluate the cell viability. AnnexinV staining and cell cycle analysis was done by flow cytometry to assess the level of apoptosis and analyze cell cycle arrest. Western blotting was performed using antibodies to analyse the impact of IB on intracellular signaling targets. Results: Myeloma cells were treated with increasing concentrations of IB for 72 hrs. Cell viability assays demonstrated that treatment of myeloma cell lines with IB resulted in an 80% decrease in cell growth using concentrations of IB between 6 and 10 uM. To determine the cause of growth inhibition and further mechanism of action, assays for apoptosis and cell cycle were performed. Annexiv V staining demonstrated that myeloma cell lines underwent brisk and rapid apoptosis. Procaspases (caspase 8, 9, 3 and PARP) were extensively cleaved from concentrations as low as 0.5uM to 10uM of IB at the 48 hour timepoint. Also, extensive DNA damage was evident based on the elevated levels of phos-p53 and GADD45 in MM.1S cells. The cell cycle profile indicated that IB induces both G2/M cell cycle arrest in MM.1S cells as well as in RPMI8226 cell lines. In addition, IB overcomes the growth advantage by cytokines IL-6 and IGF1 in MM.1S cells. Primary tumor cells obtained from myeloma patients demonstrated a significant cell killing following IB exposure. Given single agent activity, we then combined IB with other active anti-myeloma agents. Combining IB either with the proteasome inhibitor bortezomib, perifosine (an AKT inhibitor) or honokiol, a natural extract of the magnolia flower (Ishitsuka; Blood; 2005) demonstrated significant increase in the level of apoptosis favoring the combination in myeloma cells. Additionally, BAY11-7082 is an inhibitor of cytokine-induced IKB-α phosphorylation enhanced the level of apoptosis in combination with IB in the U266 myeloma cell line which is relatively less sensitive to IB, suggesting inhibition of NF-KB pathway is involved in the IB mediated myeloma cell death. Conclusion: Imipramine Blue has a potential to induce both apoptosis and G2/M cell cycle arrest in myeloma cell lines and in primary myeloma cells. The cellular response seems to be mainly mediated through pathways independent of PI3 kinase pathway, causing extensive DNA damage, cell cycle arrest, and apoptosis.

New Targets and Potential Strategy to Enhance the Anticancer Efficacy BKM-120 and BEZ235 in Lymphoma Cell Lines

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1773-1773
Author(s):  
Monica Civallero ◽  
Maria Cosenza ◽  
Stefano Sacchi
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Cycle Arrest ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Caspase 3 ◽  
Lymphoma Cell ◽  
Tubulin Polymerization ◽  
Anticancer Efficacy ◽  
Cycle Arrest

Abstract Background. The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays an important role in many physiological functions, including cell cycle progression, differentiation, survival, motility, apoptosis, protein synthesis and metabolism modification. The PI3K/AKT/mTOR signaling pathway also inhibits cell autophagy, a catabolic process involving the degradation of a cell’s own components through the lysosomal machinery. BKM120 and BEZ235 are synthetic small molecules belonging to the class of imidazo-quinolones that show preclinical activity against a range of solid and hematological malignancies. BKM-120 inhibits the catalytic subunit of class I PI3K by competitive binding to its ATP binding site, while BEZ235 is a dual class I PI3K/mTOR inhibitor. In this study we investigated the effects of BKM120 and BEZ235 on survival rate, apoptosis, signaling pathways expression, autophagy, metabolism modification, cell cycle arrest and tubulin polymerization kinetic in lymphoma cell lines. Method. Lymphoma cell lines (WSU-NHL, Jeko-1 and Karpas-299) were treated with different concentrations of BKM120 and BEZ235 (Novartis) for 24 and 48h and the IC50 values were evaluated using MTT assay. To assess for apoptosis, we used annexin V/PI staining kit/ flow cytometer analysis and Western Blot to evaluate caspase 3, 8, 9 and PARP expression. The cell cycle was performed applying PI incorporation and flow cytometer analysis. A flow cytometry based technique for the analysis of tubulin polymerization using a-tubulin staining was done to test whether the two drugs could cause a mitotic block. Western blot was utilized for phosphorylation status of protein kinases and for monitoring autophagy and metabolism. Results and conclusions. BKM120 and BEZ235 induced significant increase of apoptosis evidenced by annexin IV/PI staining and confirmed by the cleavage of caspases -3, -9 -8 and PARP. BKM120 and BEZ235 induced an up regulation of pro apoptotic protein Bim, Bax and Bad. Treatment for 24h with BKM120 and BEZ235 resulted in different effects on cell cycle. BKM120 induced an increase of G2-phase with down regulation of Cyclin D and E, and an up-regulation of Cyclin A, p21 and p27. The increase in G2-M caused by BKM120 treatment occurred in a dose dependent manner. BEZ235 induced an increase of G0/G1-phase with up regulation of Cyclin A, D, E and p21 and p27. Quantitative analysis of a-tubulins polymerization of the cell lines revealed that treatment with BKM120 induced an accumulation of mitotic cells. BKM120 and BEZ235 are inhibitors of intracellular pathways in targeting p-Akt, p- mTOR, pS6K, 4EBP1, MYC and STAT. BKM120 and BEZ235 increased the levels of type II LC3 and p62, hallmarks of autophagy, in addition to increasing caspase 3 cleavage and annexin positive cells, suggesting that the two drugs induced both apoptosis and autophagy. The combination of BKM120 and BEZ235 with chloroquine which are in clinical use, with the lysosomotropic autophagy inhibitor chloroquine demonstrating marked cooperates with inhibition of autophagy to elicit apoptosis through the intrinsic mitochondrial pathway (BAX, BIM and BAD). Treatment with BKM120 and BEZ235 induced an increase of GLUT1 and HIF-1 protein expression. GLUT1 plays a role in regulation of ROS levels in particular after BEZ235 treatment. We concluded that BKM120 and BEZ235 inhibit PI3K/AKT and mTOR signaling, induce autophagy-apoptosis, affect lymphoid cell metabolism and promote ER stress via ROS increase. Moreover, we observed that BKM120 can act as a microtubule destabilizer inducing cell cycle arrest. In these study, we highlight new targets of BKM120 and BEZ235 in addition to the known PI3K/AKT signaling pathway; these results can help to identify new potential strategies to enhance the anticancer efficacy BKM-120 and BEZ235 in lymphoma cell lines. Disclosures No relevant conflicts of interest to declare.

scholarly journals The ATM Inhibitor KU60019 Synergizes the Antineoplastic Effect of Romidepsin in Mantle Cell Lymphoma (MCL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3703-3703
Author(s):  
Luigi Scotto ◽  
Xavier Jirau-Serrano ◽  
Kelly Zullo ◽  
Michael Mangone ◽  
Jennifer E Amengual ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Cdk Inhibitor ◽  
Genetics Research ◽  
Cycle Arrest ◽  
Subcutaneous Xenograft

Abstract Introduction: Romidepsin (R), an HDAC inhibitor (HDACi) approved for the treatment of relapsed T-cell lymphoma, is thought to induce cell cycle arrest and apoptosis. Central to the block of cell proliferation is the up-regulation of the cdk inhibitor p21Cip1/Waf1. Interestingly up-regulation of p21Cip1/Waf1 has also been shown to reduce sensitivity to romidepsin. HDAC inhibitors as a class appear to activate p21Cip1/Waf1 expression via ATM. KU60019, a specific ATM inhibitor, has been shown to decrease the p21Cip1/Waf1 protein levels in a concentration dependent manner. We sought to explore the potential synergistic interaction of an ATM inhibitor with R, given the potential complementary effects around p21Cip1/Waf1. Methods: For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-GloTM. Gene expression analysis was performed via Western blot and semi-quantitative PCR assay. Apoptosis and cell cycle analysis were analyzed via Fluorescence-activated cell sorting (FACS) and Western blot. The efficacy of the compounds as single agent and combination is evaluated using a subcutaneous xenograft MCL mouse model. Results: Synergy analyses were performed using Jeko-1, Maver-1 and Z-138 cells. A synergistic cytotoxic effect was observed in all MCL cell lines when the HDACi was combined with KU60019 throughout the range of all tested concentrations. Flow cytometry analysis of all three cell lines treated with single agents and combination indicated a substantial increase in the apoptotic cell fraction when R was combined with KU60019. Furthermore protein expression analysis revealed changes in a host of proteins known to be involved in cell cycle control and apoptosis. Increased activation of the programmed cell death proteins Caspase 8 and Caspase 3 was observed upon combinations of the single agents in all three cell lines, resulting in an increased cleavage of Poly (ADP-ribose) polymerase (PARP-1) and accumulation of the DNA damage marker gammaH2AX. Finally, the abundance of the anti-apoptotic proteins Bcl-XL and BCL-2 showed a significant decrease after treatment with R plus increased concentrations of KU60019 when compared with their abundance in the presence of the single agents. Romidepsin kill tumor cells by driving premature exit from aberrant mitosis and inducing the rapid onset of apoptosis. The increased p21 protein expression upon addition of R, responsible for cell cycle arrest in the G2/M phase, was not observed when the R treatment was combined with the KU60019 suggesting that the increased apoptosis observed in the combination treatment was due to the ability of the KU60019 to affect the up-regulation of the cdk inhibitor p21Cip1/Waf1 by the HDACi. Indeed cell cycle and protein expession analysis of Jeko-1 cells treated with single agents and the combination confirmed that addition of KU60019 results in the inhibition of the p21Cip1/Waf1 induced G2/M cell cycle arrest. Western blot and transfection analysis of p21 promoter constructs indicated that activation of p21(WAF1/Cip1) transcription by HDACi in Jeko-1 cells occurs through Sp1 sites and KU60019 affected R ability to induce p21 expression at transcriptional level. A decreased in the expression level of SP1 protein upon addition of KU60019 would suggest that KU60019 affect the ability of HDACi to induce p21(WAF1/Cip1) by modulating the expression levels of SP1. A survey of the effect of R in combination with KU60019 in other tumor types derived cell lines (CTCL, DLBCL, TALL and ATLL) also shown an overall synergistic effect similar to the one observed in MCL. The potential therapeutic effect of the HDACi in combination with KU60019 is currently evaluated in a MCL subcutaneous xenograft mouse model using the Z-138 lymphoma derived cell line. Conclusions: These data support the novel concept that dual targeting of HDAC and ATM inhibition may be a synergistic and effective strategy in MCL. Disclosures Amengual: Acetylon Pharmaceuticals, INC: Consultancy, Research Funding. Deng:TG Therapeutics, Inc.: Honoraria, Research Funding; Seattle Genetics: Research Funding. O'Connor:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acetylon: Consultancy, Other: Consultancy fee; Spectrum Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb Company: Consultancy, Other: Consultancy fee; Novartis: Consultancy, Honoraria, Other: Consultancy fee; Takeda Millenium: Consultancy, Honoraria, Other: Consultancy fee, Research Funding; Seattle Genetics: Research Funding; Bayer: Consultancy, Honoraria; Mundipharma: Consultancy, Research Funding.

SF1126, a Pan-PI3K Inhibitor Has Superior Preclinical Activity to CAL-101 a PI3K Delta-Specific Inhibitor in Aggressive B-Cell Non-Hodgkin's Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2720-2720 ◽  
Author(s):  
Daruka Mahadevan ◽  
Wenqing Qi ◽  
Amy Stejskal ◽  
Laurence Cooke ◽  
Joseph R Garlich
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
B Cell ◽  
Cyclin D1 ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Specific Inhibitor ◽  
Pi3k Pathway ◽  
Cycle Arrest ◽  
G1 Cell Cycle Arrest

Abstract Abstract 2720 The PI3K pathway is constitutively active in B-cell non-Hodgkin lymphomas (B-NHL). PI3K pathway targeted therapies have focused on inhibiting mTORC1 (rapalogs) with a ∼20–48% response rate due to inactivation of mTORC1 resulting in G1 cell-cycle arrest or apoptosis. A mechanism of resistance to rapalogs is that mTORC2 is unaffected resulting in undesirable Akt activation. Strategies to block Akt up-regulation require novel agents that simultaneously block PI3K, mTORC1 and mTORC2. SF1126 is a novel pan-PI3K/mTORC1/mTORC2 inhibitor conjugated to an integrin targeted peptide RGD with potent anti-tumor activity in multiple solid tumor types. Here, we demonstrated SF1126 had potent anti-B-NHL activity and is superior to CAL-101 a PI3K delta-isoform specific inhibitor in a panel of aggressive B-NHL cell lines. Cells treated with SF1126 exhibited >90% decrease in pAkt and pGSK-3β confirming the mechanism of action of a pan-PI3K inhibitor. Moreover, SF1126 induced apoptosis in a dose and time dependent manner confirmed by flow cytometry, PARP cleavage and with an IC50 < 4μM. In contrast, CAL-101 was less active compared to SF1126 in inducing apoptosis (12% versus 25% in SUDHL-4 and 15% versus 23% in TMD-8) and cell proliferation (5.62μM versus 3.28μM SUDHL-4 and 5.31μM versus 1.47μM in TMD-8). SF1126 induced G1 cell cycle arrest at 2μM which contributes to suppression of cell proliferation. The cell cycle protein cyclin D1 is downstream of mTORC1, and the over-expression of cyclin D1 is a hallmark of mantle cell NHL (MCL). Consistent with this, cyclin D1 was significantly decreased by SF1126 compared to CAL-101. Lastly, the addition of Rituximab to SF1126 or CAL101 increased the apoptosis over single agent therapy in B-NHL cell lines. In conclusion, we demonstrate that SF1126 potently inhibits the constitutively activated PI3K/mTORC/Akt pathway in aggressive B-cell NHL cell lines with consequent suppressive effects on cell cycle progression, cell proliferation and induction of apoptosis. These findings provide a rationale for SF1126 in combination with rituximab as a novel therapeutic strategy for aggressive B-NHL and warrant early phase clinical trial evaluation [Funded by the Lymphoma SPORE 1 P50 CA 130805 01A1]. Disclosures: Garlich: Semafore Pharmaceuticals: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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