AKT Reactivation Research Articles

Integrative Modeling of Signaling Network Dynamics Identifies Cell Type-Selective Therapeutic Strategies for FGFR4-Driven Cancers.

Oncogenic FGFR4 signaling represents a potential therapeutic target in various cancer types, including triple-negative breast cancer and hepatocellular carcinoma. However, resistance to FGFR4 single-agent therapy remains a major challenge, emphasizing the need for effective combinatorial treatments. Our study sought to develop a comprehensive computational model of FGFR4 signaling and to provide network-level insights into resistance mechanisms driven by signaling dynamics. An integrated approach, combining computational network modeling with experimental validation, uncovered potent AKT reactivation following FGFR4 targeting in triple-negative breast cancer cells. Analyzing the effects of cotargeting specific network nodes by systematically simulating the model predicted synergy of cotargeting FGFR4 and AKT or specific ErbB kinases, which was subsequently confirmed through experimental validation; however, cotargeting FGFR4 and PI3K was not synergistic. Protein expression data from hundreds of cancer cell lines was incorporated to adapt the model to diverse cellular contexts. This revealed that although AKT rebound was common, it was not a general phenomenon. For example, ERK reactivation occurred in certain cell types, including an FGFR4-driven hepatocellular carcinoma cell line, in which there is a synergistic effect of cotargeting FGFR4 and MEK but not AKT. In summary, this study offers key insights into drug-induced network remodeling and the role of protein expression heterogeneity in targeted therapy responses. These findings underscore the utility of computational network modeling for designing cell type-selective combination therapies and enhancing precision cancer treatment. Significance: Computational predictive modeling of signaling networks can decipher mechanisms of cancer cell resistance to targeted therapies and enable identification of more effective cancer type-specific combination treatment strategies.

The requirement of the mitochondrial protein NDUFS8 for angiogenesis

Mitochondria are important for the activation of endothelial cells and the process of angiogenesis. NDUFS8 (NADH:ubiquinone oxidoreductase core subunit S8) is a protein that plays a critical role in the function of mitochondrial Complex I. We aimed to investigate the potential involvement of NDUFS8 in angiogenesis. In human umbilical vein endothelial cells (HUVECs) and other endothelial cell types, we employed viral shRNA to silence NDUFS8 or employed the CRISPR/Cas9 method to knockout (KO) it, resulting in impaired mitochondrial functions in the endothelial cells, causing reduction in mitochondrial oxygen consumption and Complex I activity, decreased ATP production, mitochondrial depolarization, increased oxidative stress and reactive oxygen species (ROS) production, and enhanced lipid oxidation. Significantly, NDUFS8 silencing or KO hindered cell proliferation, migration, and capillary tube formation in cultured endothelial cells. In addition, there was a moderate increase in apoptosis within NDUFS8-depleted endothelial cells. Conversely, ectopic overexpression of NDUFS8 demonstrated a pro-angiogenic impact, enhancing cell proliferation, migration, and capillary tube formation in HUVECs and other endothelial cells. NDUFS8 is pivotal for Akt-mTOR cascade activation in endothelial cells. Depleting NDUFS8 inhibited Akt-mTOR activation, reversible with exogenous ATP in HUVECs. Conversely, NDUFS8 overexpression boosted Akt-mTOR activation. Furthermore, the inhibitory effects of NDUFS8 knockdown on cell proliferation, migration, and capillary tube formation were rescued by Akt re-activation via a constitutively-active Akt1. In vivo experiments using an endothelial-specific NDUFS8 shRNA adeno-associated virus (AAV), administered via intravitreous injection, revealed that endothelial knockdown of NDUFS8 inhibited retinal angiogenesis. ATP reduction, oxidative stress, and enhanced lipid oxidation were detected in mouse retinal tissues with endothelial knockdown of NDUFS8. Lastly, we observed an increase in NDUFS8 expression in retinal proliferative membrane tissues obtained from human patients with proliferative diabetic retinopathy. Our findings underscore the essential role of the mitochondrial protein NDUFS8 in regulating endothelial cell activation and angiogenesis.

Preclinical evaluation of the CDK4/6 inhibitor palbociclib in combination with a PI3K or MEK inhibitor in colorectal cancer

ABSTRACT Background Studies have demonstrated the efficacy of Palbociclib (CDK 4/6 inhibitor), Gedatolisib (PI3K/mTOR dual inhibitor) and PD0325901 (MEK1/2 inhibitor) in colorectal cancer (CRC), however single agent therapeutics are often limited by the development of resistance. Methods We compared the anti-proliferative effects of the combination of Gedatolisib and Palbociclib and Gedatolisib and PD0325901 in five CRC cell lines with varying mutational background and tested their combinations on total and phosphoprotein levels of signaling pathway proteins. Results The combination of Palbociclib and Gedatolisib was superior to the combination of Palbociclib and PD0325901. The combination of Palbociclib and Gedatolisib had synergistic anti-proliferative effects in all cell lines tested [CI range: 0.11–0.69] and resulted in the suppression of S6rp (S240/244), without AKT reactivation. The combination of Palbociclib and Gedatolisib increased BAX and Bcl−2 levels in PIK3CA mutated cell lines. The combination of Palbociclib and Gedatolisib caused MAPK/ERK reactivation, as seen by an increase in expression of total EGFR, regardless of the mutational status of the cells. Conclusion This study shows that the combination of Palbociclib and Gedatolisib has synergistic anti-proliferative effects in both wild-type and mutated CRC cell lines. Separately, the phosphorylation of S6rp may be a promising biomarker of responsiveness to this combination.

PMON171 DRD2 Agonist Cabergoline Abolished the Escape Mechanism Induced by mTOR Inhibitor Everolimus in Pituitary Neuroendocrine Tumoral Cells

Abstract PI3K-AKT-mTOR inhibitors have been proposed as alternative therapeutic strategy for tumors resistant to conventional drugs. The mammalian target of rapamycin (mTOR) inhibitor everolimus has been shown to display antiproliferative effects on a wide spectrum of tumors. In vitro studies demonstrated that everolimus inhibited pituitary neuroendocrine tumors (PitNETs) cell growth and vitality in a subset of patients. Sensitivity to everolimus has been demonstrated to be reduced by an escape mechanism that increases AKT phosphorylation (p-AKT), leading to pro-survival pathway activation. This limited efficacy of everolimus due to AKT re-activation could be overtaken by upstream AKT phosphorylation inhibition. Given it was recently demonstrated that DRD2 mediates a reduction of p-AKT in a subset of NF-PitNETs and in rat prolactin-secreting tumor cells MMQ through a β-arrestin 2-dependent mechanism, the aim of the present work was to test the efficacy of adjuvant treatment with cabergoline in increasing sensitivity of human primary cultured NF-PitNET cells to antiproliferative effects of everolimus. In addition, to test a possible role of β-arrestin2. We found that 9 out of 14 NF-PitNETs were resistant to everolimus 1 nM, but the combined treatment with cabergoline inhibited cell proliferation in 7 out of 9 tumors (-31.4 ± 9.9%, p < 0.001 vs basal), increased p27 and reduced cyclin D3 expression. In everolimus unresponsive NF-PitNETs group, 3 h everolimus treatment determined a significant increase of p-AKT/total-AKT ratio (+2.1-fold, p < 0.01, vs basal), and this effect was reverted by cabergoline cotreatment in all tested samples. To investigate the molecular mechanism involved, we used MMQ cells as a model of everolimus escape mechanism. Indeed, 1nM everolimus did not affect MMQ cells proliferation due to an increase of p- AKT/total-AKT ratio (+1.53 ± 24%, p<0.001 vs basal), whereas cabergoline reduced cell proliferation (-22.8 ± 6.8%, p<0.001 vs basal) and AKT phosphorylation. The combined treatment of everolimus and cabergoline induced a significant reduction of both cell proliferation (-34.8 ± 18%, p<0.001 vs basal) and p-AKT/total-AKT ratio (-34.5 ± 14%, p<0.001 vs basal). To test a possible involvement of β-arrestin 2, silencing experiments were performed in MMQ cells. Our data showed that the lack of β-arrestin 2 prevented everolimus and cabergoline co-treatment inhibitory effects on both p-AKT and cells proliferation. In conclusion, these data revealed that cabergoline might overcome the everolimus escape mechanism in NF-PitNETs and tumoral lactotrophs, by inhibiting upstream AKT activation. The co-administration of cabergoline might improve mTOR inhibitors antitumoural activity, paving the way for a potential combined therapy in β-arrestin 2 expressing NF-PitNETs or other PitNETs resistant to conventional treatments. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Decrease in Tripartite Motif Containing 24 suppresses hypoxia-induced proliferation and migration of pulmonary arterial smooth muscle cells via the AKT/mammalian target of rapamycin complex 1 pathway

ABSTRACT Tripartite Motif Containing 24 (TRIM24) is an oncogenic protein and promotes proliferation in several cancer cell lines. Nevertheless, the role of TRIM24 in proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) remains to be clarified. The current study was aimed to reveal the role of TRIM24 in proliferation and migration of PASMCs during the development of pulmonary arterial hypertension (PAH). In pulmonary arteries (PAs) of chronic hypoxia-PAH (CH-PAH) mice and PASMCs challenged with hypoxia, the expression of TRIM24 was increased. Silencing TRIM24 by Trim24 short hair RNA (shTrim24) suppressed hypoxia-induced increase in Ki-67 positive PASMCs and wound-healing rate. Furthermore, hypoxia caused enhanced phosphorylation of AKT and two major effectors of mammalian target of rapamycin complex 1 (mTORC1), S6 and 4EBP1 in PASMCs. The enhancement was then attenuated after silencing TRIM24. Both restoring mTORC1 activity and AKT reactivation abolished silencing TRIM24-mediated inhibition of proliferation and migration of PASMCs. Additionally, AKT reactivation also reversed the declined phosphorylation of S6 and 4EBP1 induced by shTrim24. In conclusion, TRIM24 is involved in the excessive proliferation and migration of PASMCs after hypoxic stimulus during PAH. The mechanism of TRIM24-mediated regulation of PASMCs is partly illustrated by promoting activity of AKT/mTORC1 signaling pathway.

Aquaporin 5 Facilitates Corneal Epithelial Wound Healing and Nerve Regeneration by Reactivating Akt Signaling Pathway

Aquaporins (AQPs) are a highly conserved group of membrane proteins that play critical roles in water and small solute transport across epithelial and endothelial barriers. The aim of this study was to determine whether AQP5, a well-known water channel protein, also plays a role in corneal epithelial wound healing. AQP5 knockout (AQP5-/-) mice were generated using CRISPR/Cas9. A corneal wound healing model was established using epithelial debridement of corneas. The time to corneal epithelial and nerve regeneration was significantly delayed in the AQP5-/- mice. Reduction in Ki-67-positive cells and nerve growth factor (NGF) expression was confirmed in the AQP5-/- mice during healing. Epithelial and nerve regeneration rates were significantly increased in the AQP5-/- mice after treatment with NGF, which was accompanied by recovered levels of phosphorylated Akt. NGF treatment also improved the recovery of corneal nerve fiber density and sensitivity in the AQP5-/- mice. While the promotion of NGF induced corneal epithelial and nerve regeneration, Akt inhibitor reversed Akt reactivation. A significant impairment of corneal wound healing in the AQP5-/- mice resulted from distinct defects in corneal epithelial cell proliferation and nerve regeneration. These results provide evidence for the involvement of aquaporin in cell proliferation and suggest that AQP5 induction could be a potential therapy for accelerating the resurfacing of corneal defects.

Abstract 1098: Activation of YAP1 confers ROS1 inhibitor resistance in ROS1-rearranged lung cancer

Abstract ROS1 gene arrangements occur in approximately 1% to 2% of non-small cell lung cancers. Crizotinib, the ROS1-tyrosine kinase inhibitor (TKI), has been approved for ROS1-rearranged lung cancer based on its dramatic therapeutic effect, but cures are usually not achieved. We have been focused on relationships between Yes associated protein1 (YAP1) and resistance to molecular target drugs. YAP1, a main mediator of the Hippo pathway, promotes cell proliferation and epithelial-mesenchymal transition and is associated with drug resistance in some cancer types. We previously demonstrated that YAP1 mediates survival against alectinib therapy in anaplastic lymphoma kinase-rearranged lung cancer cells. Then, in this study, we evaluated the role of YAP1 as an initial survival mechanism from lorlatinib in ROS1-rearranged lung cancer. We established a patient-derived EZR-ROS1-rearranged lung cancer cell line (KTOR71) from pleural effusion of a patient who acquired resistance to crizotinib. Subsequently, we conducted functional analysis of YAP1 involvement in drug-resistance, using this patient-derived cell line. DNA sequencing confirmed that KTOR71 cells harbored EZR-ROS1 fusion and ROS1 S1986F mutation, which is known crizotinib resistance mutation. KTOR71 cells were sensitive to lorlatinib, while resistant to crizotinib in cell viability assay (CVA). Nuclear localization of YAP1, which is an activation marker of YAP1, was assessed using immunofluorescence staining. In KTOR71 cells, YAP1 was activated after lorlatinib exposure. Then, to evaluate functional role of YAP1 in the initial survival against lorlatinib, the genetic inhibition of YAP1 in KTOR71 cells were performed using small interfering RNA (siRNA). Silencing of YAP1 by siRNA enhanced sensitivity to lorlatinib in KTOR71 cells in CVA. Furthermore, to confirm this functional role of YAP1 in vivo, we established the KTOR71 cell-derived xenograft model. The combination therapy with lorlatinib and verteporfin, an inhibitor of YAP1-TEAD interaction, sustained tumor remission significantly compared with lorlatinib monotherapy in vivo. Finally, we found that AKT was reactivated with activation of YAP1 and silencing of YAP1 by siRNA suppressed this AKT reactivation. These results suggest that YAP1 may be a potential drug target for ROS1-rearranged lung cancer. Citation Format: Masatoshi Yamazoe, Hiroaki Ozasa, Tetsuya Ohgimoto, Kazutaka Hosoya, Hitomi Ajimizu, Tomoko Funazo, Yuto Yasuda, Takahiro Tsuji, Hironori Yoshida, Ryo Itotani, Yuichi Sakamori, Young Hak Kim, Hirai Toyohiro. Activation of YAP1 confers ROS1 inhibitor resistance in ROS1-rearranged lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1098.

Repurposing Ziyuglycoside II Against Colorectal Cancer via Orchestrating Apoptosis and Autophagy.

Effective chemotherapy drugs for colorectal cancer remain a challenge. In this research, Ziyuglycoside II (Ziyu II), exhibits considerable antitumor activity against CRC cells both in vitro and in vivo. The results showed that Ziyu II induced apoptosis through the accumulation of reactive oxygen species (ROS), which was necessary for Ziyu II to inhibit colorectal cancer cells. Intriguingly, The treatment of Ziyu II triggered complete autophagic flux in CRC cells. Inhibition of autophagy partially reversed Ziyu II-induced growth inhibition, demonstrating a cytotoxic role of autophagy in response to Ziyu II-treated. Mechanism indicated that Ziyu II-induced autophagy by inhibiting Akt/mTOR pathway. Akt reactivation partially reduced Ziyu II-induced LC3-II turnover and LC3 puncta accumulation. Especially, Ziyu II improves the sensitivity of 5-fluorouracil which is the first-line chemotherapy drug in colorectal cancer cells. This research provides novel insight into the molecular mechanism of Ziyu II’s anti-proliferation, including apoptosis and autophagy, and lays a foundation for the potential application of Ziyu II in clinical CRC treatment.

Overriding Adaptive Resistance to Sorafenib Through Combination Therapy With Src Homology 2 Domain-Containing Phosphatase 2 Blockade in Hepatocellular Carcinoma.

The survival benefit of sorafenib for patients with hepatocellular carcinoma (HCC) is unsatisfactory due to the development of adaptive resistance. Increasing evidence has demonstrated that drug resistance can be acquired by cancer cells by activating a number of signaling pathways through receptor tyrosine kinases (RTKs); nevertheless, the detailed mechanism for the activation of these alternative pathways is not fully understood. Given the physiological role of Src homology 2 domain-containing phosphatase 2 (SHP2) as a downstream effector of many RTKs for activation of various signaling cascades, we first found that SHP2 was markedly up-regulated in our established sorafenib-resistant cell lines as well as patient-derived xenografts. Upon sorafenib treatment, adaptive resistance was acquired in HCC cells through activation of RTKs including AXL, epidermal growth factor receptor, EPH receptor A2, and insulin-like growth factor 1 receptor, leading to RAS/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK), and AKT reactivation. We found that the SHP2 inhibitor SHP099 abrogated sorafenib resistance in HCC cell lines and organoid culture in vitro by blocking this negative feedback mechanism. Interestingly, this sensitization effect was also mediated by induction of cellular senescence. SHP099 in combination with sorafenib was highly efficacious in the treatment of xenografts and genetically engineered models of HCC. SHP2 blockade by SHP099 in combination with sorafenib attenuated the adaptive resistance to sorafenib by impeding RTK-induced reactivation of the MEK/ERK and AKT signaling pathways. SHP099 in combination with sorafenib may be a safe therapeutic strategy against HCC.

PTEN Inhibition Facilitates Diabetic Corneal Epithelial Regeneration by Reactivating Akt Signaling Pathway.

PurposeTo investigate the contribution of phosphatase and tensin homologue (PTEN) on the delayed epithelial regeneration and impaired Akt activation in diabetic mice.MethodsThe expression of PTEN on cornea was compared between normal and diabetic mice. The corneal epithelial and nerve regeneration rate was evaluated in diabetic mice after the treatment with PTEN small interfering RNA (siRNA), PTEN inhibitors, or Akt inhibitor. The reactivation of epithelial regeneration-related signaling, including phosphorylated (p)-Akt, p-Stat3, Sirt1, and Parkin, were assessed with Western blot and immunofluorescence staining. The effects of PTEN inhibition on cellular proliferation and migration were further evaluated in cultured mouse corneal epithelial cells.ResultsPTEN messenger RNA and protein levels exhibited up-regulation in diabetic cornea. Upon central epithelial debridement, the epithelial regeneration rate was significantly promoted in diabetic mice with the treatment of PTEN inhibition than that of vehicle control (P < 0.05), which accompanied with the recovered levels of p-Akt, p-Stat3, Sirt1, and Parkin. However, the promotion of diabetic corneal epithelial regeneration rate and Akt reactivation was completed reversed by Akt inhibitor. In vitro, PTEN inhibition promoted their migration, but not the proliferation capacity. In addition, PTEN inhibitor treatment also improved the recovery of corneal nerve fiber density and sensitivity that was impaired in diabetic mice.ConclusionsElevated PTEN expression contributes to the impaired corneal epithelial regeneration and Akt activation in diabetic mice, which can be improved with PTEN inhibition.Translational RelevanceOur study suggests that PTEN inhibition may serve as a new strategy for restoring the impaired corneal epithelial regeneration ability in patients with diabetes.

PTEN deficiency confers colorectal cancer cell resistance to dual inhibitors of FLT3 and aurora kinase A

PTEN is a tumor suppressor found mutated in many cancers. From a synthetic lethality drug screen with PTEN-isogenic colorectal cancer cells, we found that mutant-PTEN cells were resistant to dual inhibitors of FLT3 and aurora kinase-A, including KW2449 and ENMD-2076. KW2449 significantly reduced the viability of wildtype-PTEN cells causing apoptosis, while little effect was observed in mutant-PTEN counterparts. Transcriptome profiling showed that members of PI3K-AKT signaling pathway were strongly changed in cells after KW2449 treatment, indicating a potential role of the pathway in drug resistance. We found that KW2449 caused a dose-dependent, biphasic induction of AKT phosphorylation at Ser473 in mutant-PTEN cells. Co-treatment with the inhibitors of its upstream signaling completely abolished the reactivation of AKT phosphorylation by KW2449 and reversed the drug resistant phenotype. These data suggest that reactivation of AKT phosphorylation at Ser473 is a key factor to confer drug resistant phenotype of mutant-PTEN cells to the dual inhibitors and that proper drug combinations that shut down AKT reactivation is necessary for the effective treatment of mutant-PTEN cancer with the dual inhibitors in clinical settings.

Skp2-dependent reactivation of AKT drives resistance to PI3K inhibitors.

The PI3K-AKT kinase signaling pathway is frequently deregulated in human cancers, particularly breast cancer, where amplification and somatic mutations of PIK3CA occur with high frequency in patients. Numerous small-molecule inhibitors targeting both PI3K and AKT are under clinical evaluation, but dose-limiting toxicities and the emergence of resistance limit therapeutic efficacy. Various resistance mechanisms to PI3K inhibitors have been identified, including de novo mutations, feedback activation of AKT, or cross-talk pathways. We found a previously unknown resistance mechanism to PI3K pathway inhibition that results in AKT rebound activation. In a subset of triple-negative breast cancer cell lines, treatment with a PI3K inhibitor or depletion of PIK3CA expression ultimately promoted AKT reactivation in a manner dependent on the E3 ubiquitin ligase Skp2, the kinases IGF-1R (insulin-like growth factor 1 receptor) and PDK-1 (phosphoinositide-dependent kinase-1), and the cell growth and metabolism-regulating complex mTORC2 (mechanistic target of rapamycin complex 2), but was independent of PI3K activity or PIP3 production. Resistance to PI3K inhibitors correlated with the increased abundance of Skp2, ubiquitylation of AKT, cell proliferation in culture, and xenograft tumor growth in mice. These findings reveal a ubiquitin signaling feedback mechanism by which PI3K inhibitor resistance may emerge in aggressive breast cancer cells.

Redundant JAK, SRC and PI3 Kinase Signaling Pathways Regulate Cell Survival in Human Ph-like ALL Cell Lines and Primary Cells

▪Background: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk disease with a frequency of about 20% in patients of all ages. Ph-like ALL can be characterized in different groups based on their chromosomal rearrangements. The most common rearrangements involve CRLF2, ABL1, and EPOR and lead to activation of JAK kinase dependent activation of STAT5, PI3 kinase and other signaling pathways. We have previously shown that Ph-like ALL is a high-risk disease with a poor prognosis in older adults. The focus of this study is to further understand the biology of Ph-like ALL and how the cells develop drug resistance by modulating their signaling pathways to escape apoptosis.Results: We and others have identified activated STAT5 and AKT signaling in Ph-like ALL cell lines and patient samples. Interestingly, inhibition of the JAK-STAT signaling pathway using JAK-kinase inhibitors abrogated short term STA5 signaling as well as AKT signaling, but Ph-like ALL cells showed only a mild apoptotic effect or arrest in cell proliferation in vitro. Furthermore, even though xenotransplantation models of Ph-like ALL show a decrease in leukemia burden when treated with ruxolitinib, a JAK1/2 inhibitor, all mice ultimately succumb to the disease, demonstrating that Ph-like ALL does not demonstrate oncogene addiction. Based on these results we hypothesize that Ph-like ALL cells can adapt to JAK kinase inhibition by activating other signaling pathways. To test this hypothesis, we performed a time course experiment in Ph-like ALL cell lines and detected AKT reactivation shortly after JAK-kinase inhibition. In addition, we detected activation of SYK and a reduction in the PI3K negative regulator PTPN6. These observations lead to the hypothesis that Ph-like ALL may have expression and activation of the pre-B cell receptor (pre-BCR). Indeed, gene expression data from the clinical trial P9906 demonstrate that Ph-like ALL cases with CRLF2-rearrangment and JAK2 mutations express high mRNA levels of pre-B cell receptor signaling molecules, including BLNK, IGGL1, VpreB3, and ZAP70. We next hypothesized that pre-BCR mediated activation of PI3 kinase rescues Ph-like ALL cells from JAK inhibition. To study PI3K signaling we first treated Ph-like ALL cell lines and xenografts with idelalisib or INCB-50465, both selective PI3Kδ inhibitors. PI3K inhibition had only a minor effect on cell viability and again AKT was reactivated 48h later. Importantly, combining both JAK-kinase inhibition with PI3K inhibition, prevented AKT reactivation and had strong synergistic effect on cell viability and cell proliferation in vitro. Xenotransplantation models of Ph-like ALL with ruxolitinib and INCB-50465 showed a significantly lower leukemia burden as compared to the single agent treatments but disease eradication was not achieved. Pre-BCR signaling can also activate SRC family kinases and we hypothesized that CRLF2-rearanged Ph-like ALL might be sensitive to SRC inhibition as well. Strikingly, combining JAK2, PI3K, and SRC inhibition further reduced the cell viability by one log scale compared to the JAK2/PI3K inhibitor treated samples and less than 0,2% viable cells were detected, while other ALL subtypes were not affected. In vivo validation of treatment strategy is pending.Clinical relevance: While children with ALL currently have a 5-year survival rate of 90%, adults, especially those over the age of 40, have a very poor clinical outcome with survival rates of about 40%. Adults often do not tolerate high doses of chemotherapy regimens and are therefore in clinical need of alternative treatment regimens. We therefore combined JAK1/2 and PI3K inhibitory treatments with dasatinib, a frequently-used tyrosine kinase inhibitor that is well tolerated by older patients. Strikingly, the combinatory treatment had a strong effect on cell viability and represents so far, the first study using specific signaling inhibitors combined in Ph-like ALL.Conclusion: These findings identify that Ph-like ALL are not oncogene addicted like Ph+ ALL. While BCR-ABL1 activates multiple signaling pathways that can be inhibited with ABL kinase inhibitors, Ph-like ALL need to be treated with multiple inhibitors to induce the same effect. Here we show that a combination of JAK, PI3K and SRC-kinase inhibitors is needed to prevent reactivation of signaling pathways and to induce an apoptotic effect that may be clinically relevant. DisclosuresHurtz:Incyte: Research Funding. Tasian:Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences, Inc.: Research Funding. Ruggeri:Incyte Corporation: Employment, Equity Ownership. Stubbs:Incyte Corporation: Employment, Equity Ownership. Perl:Daiichi Sankyo: Consultancy; Asana Biosciences: Other: Scientific advisory board; Arog Pharmaceuticals: Consultancy; Novartis: Other: Advisory Board; Seattle Genetics: Other: Advisory board; Astellas: Consultancy; Pfizer: Other: Advisory Board; Actinium Pharmaceuticals: Other: Scientific Advisory Board. Carroll:Incyte Pharmaceuticals: Research Funding; Astellas Pharmaceuticals: Research Funding.

Abstract 2618: Oncogenic mutant KRAS modulates CREB activation through MEK-ERK and AKT signaling in pancreatic cancer

Abstract Introduction: Cyclic AMP (cAMP) response element binding (CREB) overexpression in pancreatic ductal adenocarcinoma (PDAC) is associated with poor outcome, however, the mechanism(s) driving CREB overexpression in PDAC are not fully understood. We investigated the association of CREB activation with oncogenic KRAS, MEK-ERK and AKT signaling pathways. Experimental procedure: Mouse lines derived from the Ptf1aCre/+; LSL-KrasG12D/+ (K518), Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT), LSL-KrasG12D/+;Pdx1Cre/+ (PanIN) and LSL-KrasG12D/+; Trp53R172H/+;Pdx1Cre/+ (PDA) mouse models of PDAC, human immortalized pancreatic ductal epithelial lines (HPDE6-E6E7 or H6c7, HPNE E6/E7 and HPNE E6/E7/KRAS) were immunoblotted for phospho-CREB (pCREB) and total CREB expression. KRAS mutant PDAC cell lines and CREB shRNA flank xenografts (PKT GEMM and nude mice) were treated with CREB (ICG-001), MEK (AZD6244) and AKT (MK-2206) inhibitors. The effects on downstream signaling targets were interrogated with cell cycle, apoptosis, survival and anchorage-independent growth analysis. Human CREB shRNA cells were treated with AZD6244 and MK-2206 to confirm the molecular mechanism of the CREB inhibition together with MEK and AKT. Results: The expression of pCREB was higher in cells with KRAS mutation. MEK inhibition resulted in activation of AKT, while combined inhibition of CREB and MEK prevented AKT reactivation. Treatment with the combination of MEK and CREB inhibitors significantly decreased tumorigenic potential and increased cell apoptosis. Combined MEK, AKT and CREB inhibition synergistically enhanced these effects further, more so in KRAS mutant cell lines. To explore the relationship of CREB, MEK and AKT signaling in vivo, PKT mice were treated with their respective inhibitors individually and in combination. Either CREB/MEK or CREB/AKT two drug combinations significantly extended the median survival compared with individual agents. Lysates from MEK and AKT inhibited tumors showed decreased phosphorylation of CREB, confirming that CREB is activated through both, MEK and AKT signaling in vivo. Conclusions: Our study demonstrates that oncogenic KRAS activation enhances the expression of CREB through MEK and AKT signaling. CREB inhibition results in increased sensitivity to MEK and AKT targeted therapy in KRAS mutant PDACs. Citation Format: Jason Castellanos, Supriya Srinivasan, Kumar Honnenahally, Chanjuan Shi, Michael VanSaun, David Robbins, Nipun Merchant, Nagaraj Nagathihalli. Oncogenic mutant KRAS modulates CREB activation through MEK-ERK and AKT signaling in pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2618.

Reactivation of ERK and Akt confers resistance of mutant BRAF colon cancer cells to the HSP90 inhibitor AUY922.

Oncogenic mutations of BRAF occur in approximately 10% of colon cancers and are associated with their resistance to clinically available therapeutic drugs and poor prognosis of the patients. Here we report that colon cancer cells with mutant BRAF are also resistant to the heat shock protein 90 (HSP90) inhibitor AUY922, and that this is caused by rebound activation of ERK and Akt. Although AUY922 triggered rapid reduction in ERK and Akt activation in both wild-type and mutant BRAF colon cancer cells, activation of ERK and Akt rebounded shortly in the latter leading to resistance of the cells to AUY922-induced apoptosis. Reactivation of ERK was associated with the persistent expression of mutant BRAF, which, despite being a client of HSP90, was only partially degraded by AUY922, whereas reactivation of Akt was related to the activity of the HSP90 co-chaperone, cell division cycle 37 (CDC37), in that knockdown of CDC37 inhibited Akt reactivation in mutant colon cancer cells treated with AUY922. In support, as a HSP90 client protein, Akt was only diminished by AUY922 in wild-type but not mutant BRAF colon cancer cells. Collectively, these results reveal that reactivation of ERK and Akt associated respectively with the activity of mutant BRAF and CDC37 renders mutant BRAF colon cancer cells resistant to AUY922, with implications of co-targeting mutant BRAF and/or CDC37 and HSP90 in the treatment of mutant BRAF colon cancers.

RACK1 overexpression is linked to acquired imatinib resistance in gastrointestinal stromal tumor.

Although treatment with imatinib, which inhibits KIT and PDGFR, controls advanced disease in about 80% of gastrointestinal stromal tumor (GIST) patients, resistance to imatinib often develops. RACK1 (Receptor for Activated C Kinase 1) is a ribosomal protein that contributes to tumor progression by affecting proliferation, apoptosis, angiogenesis, and migration. Here, we found that c-KIT binds to RACK1 and increases proteasome-mediated RACK1 degradation. Imatinib treatment inhibits c-KIT activity and prevents RACK1 degradation, and RACK1 is upregulated in imatinib-resistant GIST cells compared to non-resistant parental cells. Moreover, Erk and Akt signaling were reactivated by imatinib in resistant GIST cells. RACK1 functioned as a scaffold protein and mediated Erk and Akt reactivation after imatinib treatment, thereby promoting GIST cell survival even in the presence of imatinib. Combined inhibition of KIT and RACK1 inhibited growth in imatinib-resistant GIST cell lines and reduced tumor relapse in GIST xenografts. These findings provide new insight into the role of RACK1 in imatinib resistance in GIST.

RICTOR involvement in the PI3K/AKT pathway regulation in melanocytes and melanoma.

Several studies have highlighted the importance of the PI3K pathway in melanocytes and its frequent over-activation in melanoma. However, little is known about regulation of the PI3K pathway in melanocytic cells. We showed that normal human melanocytes are less sensitive to selective PI3K or mTOR inhibitors than to dual PI3K/mTOR inhibitors. The resistance to PI3K inhibitor was due to a rapid AKT reactivation limiting the inhibitor effect on proliferation. Reactivation of AKT was linked to a feedback mechanism involving the mTORC2 complex and in particular its scaffold protein RICTOR. RICTOR overexpression in melanocytes disrupted the negative feedback, activated the AKT pathway and stimulated clonogenicity highlighting the importance of this feedback to restrict melanocyte proliferation. We found that the RICTOR locus is frequently amplified and overexpressed in melanoma and that RICTOR over-expression in NRAS-transformed melanocytes stimulates their clonogenicity, demonstrating that RICTOR amplification can cooperate with NRAS mutation to stimulate melanoma proliferation. These results show that RICTOR plays a central role in PI3K pathway negative feedback in melanocytes and that its deregulation could be involved in melanoma development.

Abstract A34: Therapeutic potential of the novel mTOR inhibitor Torin-2 to overcome AKT reactivation in B-precursor acute lymphoblastic leukemia (B-pre ALL).

Abstract B-precursor acute lymphoblastic leukemia (B-pre ALL) is characterized by malignant proliferation and accumulation of early lymphoid precursor cells in the bone marrow, blood and lymphoid organs, due to acquired mutations in early B-cells. Around 20% of children and the majority of adults relapses after the treatment and the poor outcome following relapse did not significantly changed over the past twenty years. For this reason, many research efforts are currently devoted to the development of targeted therapies to limit side effects of chemotherapy and to increase treatment efficacy for poor prognosis patients. Constitutively active phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling is a common feature of leukemia patients, where it negatively influences response to therapeutic treatments. Thus, targeting the PI3K/AKT /mTOR pathway is an attractive therapeutic strategy. Rapamycin, an allosteric inhibitor of mTORC1, acutely inhibits mTORC1 but not mTORC2 activity. Prolonged suppression of mTORC1 also results in disruption of a negative feedback loop and consequently results in hyperphosphorylation of Akt through activation of IRS1 and PI3K. mTOR inhibitors are currently in clinical trial for a range of hematological malignancies including ALL, mostly in combination with conventional chemotherapy. RAD001, a mTORC1 inhibitor, has been shown to affect cell cycle progression and survival with a potentially relevant therapeutic efficacy. However, prolonged inhibition of mTORC1 often leads to AKT re-phosphorylation at Ser473which may counteract RAD001 activity. A new class of ATP competitive mTOR inhibitors, such as Torin-2, have revealed that this new class of inhibitors potently targets mTORC1 and mTORC2. Together, these features have generated hope that the new generation of ATP-competitive mTOR inhibitors will exhibit broader clinical efficacy relative to the rapalogs. We therefore hypothesized that dual inhibition of mTORC1 and mTORC2 by Torin-2 would provide a superior outcome in ALL as compared to inhibition of mTORC1 alone with RAD001 in B-pre-ALL. We tested the capability of Torin-2 to prevent AKT reactivation after mTORC1 and mTORC2 inhibition. Furthermore we explored if dual targeting of mTORC1 and AKT may achieve results similar to those obtained with Torin-2 alone. We used a panel of B-pre-ALL to test the drugs by MTT Assay and Western Blotting after different time exposures of the cells. Cell cycle, apoptosis and autophagy were analyzed by flow cytometry, Western Blotting and fluorescent staining. In all the B-pre-ALL cell lines Torin-2 showed a powerful cytotoxic activity, inhibiting the growth of each cell line in a dose-dependent manner, with an IC50 in the nanomolar range as assessed by MTT assays. The major proteins along the PI3K/AKT/mTOR signaling pathway where heavily dephosphorylated after 2 hrs of drug exposure. This inhibition lasted up to at least 48 hrs at variance to RAD001, that already after 24 hrs was unable to prevent AKT reactivation. However the association of RAD001 with MK-2206, an allosteric AKT inhibitor, prevented AKT reactivation and reached a significant cytotoxicity. Our data suggest an interesting cytotoxicity of Torin-2 in B-pre-ALL acting on both mTORC1 and mTORC2 as assessed by their substrate inhibition. Torin-2 alone suppresses feedback activation of PI3K/AKT, whereas RAD001 requires the addition of MK-2206 to achieve the same efficacy. These two pharmacological options targeting PI3K/Akt/mTOR at different points of the signaling pathway cascade might represent a new therapeutic potential for treatment of B-pre-ALL patients. Citation Format: Alice Cani, Carolina Simioni, Alberto M. Martelli, Giorgio Zauli, Silvano Capitani, Luca M. Neri. Therapeutic potential of the novel mTOR inhibitor Torin-2 to overcome AKT reactivation in B-precursor acute lymphoblastic leukemia (B-pre ALL). [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A34.

Abstract 4198: mTORC2 directs breast morphogenesis through Rictor-dependent PKCα/Rac1 signaling independent of Akt

Abstract Akt phosphorylation is a major driver of cell survival, motility, and proliferation in development and disease, causing increased interest in upstream regulators of Akt like mTOR complex 2 (mTORC2). We used genetic disruption of Rictor to impair mTORC2 activity in mouse mammary epithelia, which decreased Akt phosphorylation, ductal length, secondary branching, cell motility, and cell survival. These effects were recapitulated with a pharmacological dual inhibitor of mTORC1/mTORC2, but not upon genetic disruption of mTORC1 function via Raptor knock-out. Surprisingly, Akt re-activation was insufficient to rescue survival, branching, or motility of mTORC2-impaired mammary epithelial cells (MECs). However, activation of the mTORC2 substrate protein kinase C (PKC)-α fully rescued branching, invasion, and survival of MECs after genetic or pharmacological mTORC2 inhibition. PKCα-mediated signaling through the small GTPase Rac1 was necessary for mTORC2-dependent mammary morphogenesis, revealing a novel role for Rictor/mTORC2 in survival and motility of untransformed MECs through an Akt-independent, PKCα/Rac1-dependent mechanism. Ongoing analyses of mammary specific Rictor-deletion in genetically engineered mouse models of breast cancer support a key role for mTORC2 in tumor progression and metastasis. Citation Format: Dana M. Brantley-Sieders, Meghan M. Morrison, Donna J. Hicks, Rebecca S. Cook. mTORC2 directs breast morphogenesis through Rictor-dependent PKCα/Rac1 signaling independent of Akt. [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 4198. doi:10.1158/1538-7445.AM2015-4198

Abstract 744: Transactivation of HER3 via heterodimerization with epidermal growth factor receptor (EGFR) or insulin-like growth factor 1 receptor (IGF-1R) contributes to adaptive resistance to NVP-BKM120 in non-small cell lung cancer (NSCLC) and squamous cell carcinom

Abstract Purpose: To investigate adaptive resistant mechanisms to NVP-BKM120, a pan PI3K inhibitor, in NSCLC and SCCHN cells. Experimental Design: A panel of 16 NSCLC and 11 SCCHN cell lines, including PDX01 cell line developed from a biopsy sample of SCCHN patient showing resistance to NVP-BKM120, was used. The growth inhibitory effect of NVP-BKM120 was assessed using MTT assay and colony formation assays. Receptor tyrosine kinases (RTKs) array was used to identify candidate RTKs associated with adaptive resistance to NVP-BKM120. Signaling pathways were analyzed by immunoprecipitation, western blot, and siRNAs transfection. Results: Treatment of NVP-BKM120 showed the limited antitumor effects with IC50 of 0.5 to 1.5 μM in a panel of NSCLC and SCCHN cells. The sensitivity to BKM120 was not associated with genetic alterations of PI3K pathway (e.g. PIK3CA mutation). Although AKT phosphorylation decreased markedly upon NVP-BKM120 treatment, AKT activity was rapidly restored following 4 hrs of drug treatment. Moreover, using western blot analysis and RTK array, upregulation of HER3 expression and activity from 1 hr up to 72 hrs of NVP-BKM120 treatment was observed in the majority of NSCLC and SCCHN cells. The knockdown of HER3 with NVP-BKM120 resulted in a synergistic antitumor effect with marked suppression of NVP-BKM120-triggered AKT reactivation. Interestingly, the phosphorylation of kinase-inactive HER3 at 4 hrs of NVP-BKM120 treatment was completely suppressed by gefitinib, a selective EGFR TKI, or EGFR knockdown while gefitinib or EGFR knockdown failed to inhibit HER3 phosphorylation at 24 hrs of NVP-BKM120 treatment. In line with these results, immunoprecipitation assays demonstrated the NVP-BKM120-enhanced interaction of HER3 and EGFR only at 4 hrs with recruitment of p85 and c-SRC, leading to the transient phosphorylation of EGFR, specifically at Y845 and Y1173 sites. These results suggested that HER3 activation was driven by dimerization with other RTKs at later time points after drug treatment. In addition, OSI-906, a selective inhibitor of IGF-1R, and IGF-1R-specific siRNA also reduced NVP-BKM120-induced HER3 phosphorylation, resulting in a synergistic antitumor effect in a panel of NVP-BKM120 resistant cells. Conclusions: Transactivation of HER3 via EGFR or IGF-1R attenuates the effectiveness of NVP-BKM120 in multiple NSCLC and SCCHN cells. Our results suggest that either targeting HER3 directly or indirectly by inhibiting transactivation partners, such as EGFR or IGF-1R, may be potential therapeutic options to overcome adaptive resistance to NVP-BKM120. Note: This abstract was not presented at the meeting. Citation Format: Miran Yun, Jinyoung Sohn, Byoung Chul Cho. Transactivation of HER3 via heterodimerization with epidermal growth factor receptor (EGFR) or insulin-like growth factor 1 receptor (IGF-1R) contributes to adaptive resistance to NVP-BKM120 in non-small cell lung cancer (NSCLC) and squamous cell carcinom [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 744. doi:10.1158/1538-7445.AM2015-744