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The present studies sought to determine whether the anti-folate pemetrexed (Alimta) and the sphingosine-1-phosphate receptor modulator FTY720 (Fingolimod, Gilenya) interacted to kill tumor cells. FTY720 and pemetrexed interacted in a greater than additive fashion to kill breast, brain and colorectal cancer cells. Loss of p53 function weakly enhanced the toxicity of FTY720 whereas deletion of activated RAS strongly or expression of catalytically inactive AKT facilitated killing. In contrast, expression of activated forms of AKT, p70 S6K and mTOR or inhibition of JNK and p38 MAPK suppressed the toxic interaction between FTY720 and pemetrexed. Indeed, combined drug exposure reduced the activity of AKT, p70 S6K and mTOR and activated JNK and p38 MAPK. Expression of activated forms of AKT, p70 S6K and mTOR or inhibition of JNK and p38 MAPK suppressed the toxic interaction between FTY720 and pemetrexed. Treatment of cells with FTY720 and pemetrexed increased the numbers of autophagosomes in cells that correlated with increased LC3 processing and increased p62 levels. Knock down of ATG5 or Beclin1 suppressed autophagosome formation and cell killing by the drug combination. Sorafenib was previously shown to potently interact with pemetrexed to kill tumor cells. Sorafenib significantly enhanced the lethality of [FTY720 + pemetrexed] treatment in vitro and in vivo. Collectively our findings argue that pemetrexed, sorafenib and FTY720 could be a novel adjunct modality for breast cancer treatment. Note: This abstract was not presented at the meeting. Citation Format: Jane L. Roberts, Seyedmehrad Tavallai, Andrew Poklepovic, Laurence Booth, Paul Dent. Gilenya and Alimta interact to kill breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3503. doi:10.1158/1538-7445.AM2015-3503

Detachment of cells from the extracellular matrix (ECM) results in a form of cell death referred to as anoikis. Anoikis resistance (AR), frequently acquired by malignant cells, is required for tumor metastases. The present study focused on identifying the mechanisms by which drug sensitivity is restored in previously resistant AR breast cancer cells. AR breast cancer cells display reduced expression of the toxic BH3 domain proteins BAX, BAK, NOXA and PUMA. In contrast, expression of the protective BCL-2 family proteins BCL-XL and MCL-1 was increased. It has previously been noted that AR cells display resistance to several currently available anti-tumor cell therapies, including a combinational approach with Lapatinib (ERBB1/2 inhibitor) and Obatoclax (MCL-1 inhibitor, exhibiting reduced autophagic flux, despite similarly exhibiting increased levels of LC3II processing. Knockdown of MCL-1 and BCL-XL in AR cells resulted in apoptosis to a greater extent than in their wild-type counterparts. Pre-treatment of AR cells with Valproate (histone deacetylase inhibitor (HDACIs)); for 24hrs increased the levels of toxic BH3 domain proteins whilst simultaneously reducing levels of MCL-1 and re-sensitising AR cells to therapy-mediated cell death. In vivo, pre-treatment of AR breast tumors in the brain with valproate restored chemo-sensitivity and prolonged animal survival. These data argue that pre-treatment with HDACIs may provide a mechanism to enhance the anti-tumor effect of chemotherapy in AR breast cancer cells. Citation Format: Nichola A. Cruickshanks, Hossein A. Hamed, Larry A. Booth, Seyedmehrad Tavallai, G B. Sajithlal, Steven Grant, Andrew Poklepovic, Paul Dent. Histone deacetylase inhibitors restore toxic BH3 domain protein expression in anoikis-resistant mammary and brain cancer stem cells, thereby enhancing the response to anti-ERBB1/ERBB2 therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 796. doi:10.1158/1538-7445.AM2014-796

The present study aimed to determine whether the multikinase inhibitors, sorafenib/regorafenib, worked in conjunction with PI3K/AKT inhibitors to enhance tumor cell death. It has been noted that combination of sorafenib/regorafenib with a PI3K inhibitor acetic acid (PX-866) resulted in tumor cell death, in a greater than additive fashion, in liver, colorectal, lung, breast, kidney and brain cancer cells. Similar data was obtained using AKT inhibitors perifosine and MK2206. Furthermore, even cells lacking PTEN remained as sensitive to this combinational approach as cells expressing PTEN. PX-866 treatment abolished AKT/GSK3 phosphorylation, with tumor cell death correlating with reduced activity of AKT and mTOR. Expression of activated AKT and to a lesser extent activated mTOR reduced drug combination lethality. Expression of B-cell lymphoma-extra large or dominant negative caspase 9, but not cellular FLICE-inhibitory protein short, protected cells from the drug combination. Treatment of cells with PX-866 increased protein levels of p62, LAMP2 and LC3/LC3II which correlated with the resulting increase in LC3-GFP vesicle numbers. Exposure of PX-866 treated cells to sorafenib reduced p62 and LAMP2 levels, decreased the ratio of LC3 to LC3II and reduced LC3-GFP vesicle levels. Knockdown of Beclin1 or ATG5 suppressed drug toxicity by ∼40%. In vivo, combination of either sorafenib and PX-866 or regorafenib and MK2206 cooperated to suppress the growth of established HuH7 and HCT116 tumors, respectively. Collectively our data demonstrates that the combination of sorafenib family kinase inhibitors with inhibitors of the PI3K/AKT pathway kills tumor cells in vitro and in vivo. Citation Format: Larry A. Booth, Nichola A. Cruickshanks, G B. Sajithlal, Hossein A. Hamed, Seyedmehrad Tavallai, J Syed, Steven Grant, Andrew Poklepovic, Paul Dent. Sorafenib/regorafenib and phosphatidyl inositol 3 kinase/thymoma viral proto-oncogene inhibition interact to kill tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2690. doi:10.1158/1538-7445.AM2014-2690