S6 Inhibition Research Articles

Invivo anti-cancer activity of 10-methyl-aplog-1, a simplified analog of aplysiatoxin, and its possible signaling pathway associated with G1 arrest

Naturally occurring protein kinase C (PKC) activators such as phorbol esters, teleocidins, and aplysiatoxins, have the potential to become anti-cancer agents, since they are anti-proliferative against specific cancer cell lines in vitro. However, their potent tumor-promoting and proinflammatory activities have hampered their clinical uses. Recently, we developed 10-methyl-aplog-1 (1), a simplified analog of tumor-promoting debromoaplysiatoxin (DAT), which retained anti-proliferative activity comparable to DAT, but induced neither tumorigenesis nor inflammation on mouse skin. Our previous study suggested that PKCα and δ were involved in the cell line-selective anti-proliferative activity of 1, but the downstream signaling of PKC isozymes remained unknown. In this study, we confirmed that 1 inhibited the growth of three aplog-sensitive cancer cell lines (NCI–H460, HCC-2998, and HBC-4) without severe side effects in mice xenograft models. In addition, in vitro analysis using A549, one of the aplog-sensitive cell lines in vitro, revealed that PKCα induced PP2A-mediated attenuation of the Akt/S6 signaling axis. Since S6 inhibition in A549 was reported to result in G1 arrest, this pathway could be involved in the PKCα-dependent anti-proliferative activity of 1.

DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression.

Myeloproliferative neoplasms (MPNs) exhibit a propensity for transformation to secondary acute myeloid leukemia (sAML), for which the underlying mechanisms remain poorly understood, resulting in limited treatment options and dismal clinical outcomes. Here, we performed single-cell RNA sequencing on serial MPN and sAML patient stem and progenitor cells, identifying aberrantly increased expression of DUSP6 underlying disease transformation. Pharmacologic dual-specificity phosphatase (DUSP)6 targeting led to inhibition of S6 and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling while also reducing inflammatory cytokine production. DUSP6 perturbation further inhibited ribosomal S6 kinase (RSK)1, which we identified as a second indispensable candidate associated with poor clinical outcome. Ectopic expression of DUSP6 mediated JAK2-inhibitor resistance and exacerbated disease severity in patient-derived xenograft (PDX) models. Contrastingly, DUSP6 inhibition potently suppressed disease development across Jak2V617F and MPLW515L MPN mouse models and sAML PDXs without inducing toxicity in healthy controls. These findings underscore DUSP6 in driving disease transformation and highlight the DUSP6-RSK1 axis as a vulnerable, druggable pathway in myeloid malignancies.

Abstract B074: Sapanisertib in combination with trametinib in glioblastoma models enhances target inhibition and antitumor activity

Abstract Objective: To determine the biological and signaling effects of dual-pathway inhibition with mTORC1/2 and MEK inhibitors in glioblastoma neurosphere cell lines. Background: The PI3K/mTOR signaling pathway is frequently dysregulated in glioblastoma (GBM), making it the focus of active scientific and drug development efforts in brain tumors. Unfortunately, small molecule targeted therapies against EGFR and its downstream effector pathways, while promising in the pre-clinical setting, have been disappointing in glioma clinical trials. The mTOR pathway target ribosomal protein S6 (RPS6) can also be activated by the ERK pathway target p90-RSK. We therefore tested the hypothesis that small molecule inhibition of both ERK and mTOR signaling pathways would result in enhanced anti-tumor activity. Methods: Seven glioblastoma neurosphere lines were tested for sensitivity to the ATP-competitive mTORC1/2 inhibitor sapanisertib (MLN0128) alone or in combination with the allosteric MEK1/2 inhibitor trametinib (GSK1120212) using cell growth assays and immunoblots as indicated. Results: The dual mTORC1/2 inhibitor sapanisertib inhibits mTOR signaling and cell growth in all GBM neurosphere lines, but only partially inhibits phosphorylation of the downstream target ribosomal protein S6 (phospho-RPS6) in these models. The MEK inhibitor, trametinib, inhibited ERK phosphorylation in all neurosphere lines, though phosphorylation of downstream targets and cell growth were inhibited to varying degrees. Inhibition of RPS6 activation was dose-dependent and correlated with cell line sensitivity when treated with trametinib but not with sapanisertib. In combination, sapanisertib plus trametinib showed more profound inhibition of phospho-RPS6, as well as enhanced growth inhibition and induction of apoptosis in sensitive neurosphere lines. Conclusions: The combination of sapanisertib and trametinib may have significant antitumor activity in glioblastoma neurospheres by enhanced inhibition of S6, which serves as the convergence point between these effector pathways. Citation Format: Karisa C Schreck, Amy Allen, Jiawan Wang, Christine A Pratilas. Sapanisertib in combination with trametinib in glioblastoma models enhances target inhibition and antitumor activity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B074. doi:10.1158/1535-7163.TARG-19-B074

Abstract 3511: The mechanism of antitumor effect of metformin forgemcitabine-resistant pancreatic adenocarcinoma

Abstract Metformin (MET) is the first-line treatment for type 2 diabetes mellitus. Several epidemiological studies have reported anti-cancer effects of MET, including against pancreatic ductal adenocarcinoma (PDAC), which mainly acts through induction of AMP activated protein kinase (AMPK). Gemcitabine (GEM) has become the standard chemotherapy for PDAC but tolerance to GEM has become a burdensome issue. We evaluated the anti-tumor effects of MET for GEM-resistant PDAC in a xenograft mouse model. For this in vivo study, BxG30 (the cell line for GEM-resistant PDCA) was implanted into both flanks of female BALB/c nude mice. Mice were divided into four groups: (i) control (no treatment); (ii) the GEM-treated group (100 mg/kg); (iii) the MET-treated group (600 mg/kg); and (iv) the combined treatment group (G+M). Mice were fed for 4 weeks. Estimated tumor volumes and body weights were measured each week. Treatments were initiated 2 weeks after implantation. MET was administrated orally once per day. GEM was given by intraperitoneal injection once per week. Compared with the control, the final tumor volumes were decreased significantly only in the G+M group. [SCW1] The treated control ratio (T/C%) was calculated: GEM, 80.2%; MET, 54.0%; G+M, 47.2%. The anti-tumor effect of GEM for BxG30 was clearly limited[SCW2] . MET group showed satisfactory anti-tumor effects, but T/C% was <50% only in the G+M group. This result revealed that combination therapy had excellent anti-tumor effects even for GEM-resistant PDAC. The phosphorylation of ribosomal proteins S6 and 4E-BP, important targets of the mammalian target of rapamycin (mTOR) signaling pathway, was surveyed by western blot analysis. Western blot analysis showed inhibition of S6 and 4E-BP phosphorylation by co-incubation with MET, but not with GEM. Hypoxia-inducible factor 1 (HIF-1) is the one of the most important target of mTOR signaling pathway influencing tumor cell to progress, thus the expression level of HIF-1 was evaluated by western blot analysis as well. The results showed significant inhibition of HIF-1 expression by MET treatment, but not by GEM incubated under hypoxia (95%N2, 5%O2). Then the production of VEGF was evaluated by ELISA under hypoxia. The result showed suppression of VEGF production by MET treatment, but not by GEM. Our data showed that MET develops a different anti-tumor effect from GEM by suppression of mTOR-HIF-1 signaling. These results are of great clinical interest and reveal the potential of another anti-tumor agent for treatment of PDAC. Citation Format: Keiichi Suzuki, Osamu Takeuchi, Yukio Suzuki. The mechanism of antitumor effect of metformin forgemcitabine-resistant pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3511.

Abstract 3047: Sensitization of prostate cancer to chemo-radiation by ribosomal protein S6 inhibition

Abstract Radiation Therapy (RT) is curative for most localized prostate cancer (PCA) patients, but is limited by dose-related toxicities and radioresistance. Administration of Nexrutine® (Nx), an inexpensive supplement from Phellodendron amurense bark extract prior to RT inhibited progression to poorly differentiated carcinoma in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. However, the precise molecular mechanism underlying Nx-mediated tumor growth inhibitory effects in combination with RT is unclear. Using global transcriptome profiling coupled with GeneSpring analysis, we have identified significant modulation of number of signaling pathways including genes involved in mTOR and NFκB signaling. Based on these data, we hypothesized that downregulation of ribosomal protein S6 (rpS6) sensitizes prostate tumor to overcome radioresistance. PCA cell lines had higher levels of total and p-rpS6 compared to non-tumorigenic prostate cell lines. Using clonogenic assays, we observed that pretreatment of Nx for 8h could potentiate the effect of low dose RT in LNCaP (AR positive with WT p53), PC-3 (AR negative & p53 null) and PC-3 AR (AR expressing & p53 null) cells. The combinatorial effect depicted strong synergism and had similar effects as high dose RT. Notably, we observed increased activation of Akt/mTOR/NFκB signaling molecules after treatment of PC-3 cells with RT, which was either abrogated (p-rpS6, p-NFκB & p-p70S6K) or decreased (p-Akt) in cells pretreated with Nx prior to RT. Also, Nx pretreatment inhibited the levels of HIF1-α and CyclinD1, which are downstream targets of rpS6. Using immunofluorescence assay, we saw that the combination treatment increased the γH2AX foci compared to 2 Gy alone, indicating increased DNA damage. This is associated with increased protein levels of key G2/M regulatory proteins including Wee-1, p-cdc2 levels and decreased Chk1, p-cdc25C resulting in prolonged RT-induced G2/M arrest. The combination treatment also caused a significant increase in apoptosis, which was evidenced by increased cleaved PARP levels, subG1 levels and Annexin/PI staining. Strikingly, knockdown of rpS6 sensitized PC-3 cells to RT and reversed the observed effects of Nx, indicating the importance of rpS6 in mediating these changes. Remarkably, p-mTOR, p70S6K, NFκB, Ki67 & Cyclin D1 were decreased in the prostate tissue of mice receiving the combination compared to RT alone. Recently, a phase 0/1 clinical trial of Nx in PCA patients receiving RT decreased PSA levels and was well-tolerated. Interestingly, tumor tissue from Nx treated patients showed decreased rpS6 staining intensity compared to the normal tissue. Collectively, our data suggests that Nx has radiosensitizing effect in a range of PCA cell lines and could prevent progression to advanced PCA by inhibiting rpS6. Supported by NCCAM (R01 AT-007448) & VA-MERIT Award (I01 BX 000766) Citation Format: Suleman S. Hussain, Carolina B. Livi, Nikos Papanikolaou, Daniel C. Chan, Rita Ghosh, Addanki P. Kumar. Sensitization of prostate cancer to chemo-radiation by ribosomal protein S6 inhibition. [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 3047.

Utilizing camelid-derived single domain antibodies to enhance immunotherapy of ovarian cancer

growth, and cancer cells are well known to be “glutamine-addicted.” Glutamine is converted by glutaminase to glutamate, and glutamate is converted by glutamate dehydrogenase (GDH) to α-ketoglutarate, which enters the tricarboxylic acid cycle for the production of adenosine triphosphate (ATP). In addition, glutamine generates the antioxidant glutathione, which removes reactive oxygen species (ROS) and regulates critical cell signaling pathways. Our objective was to explore the effects of glutamine on ovarian cancer (OC) cell growth and metabolism. Methods: We used the OC cell lines HEY, SKOV3, and IGROV1 and assessed dell proliferation by MTT assay. Cell cycle progression and apoptosis were evaluated by Cellometer. Western immunoblotting was performed to evaluate the effects of glutamine on downstream targets of the mTOR and MAPK pathways and proteins related to cellular stress. Glucose uptake, GDH activity, lactate, ATP, and ROS production were all assessed by enzyme-linked immunosorbent assay. The effect of mTOR pathway inhibition on glutaminestimulated cell proliferation was assessed by 1) treatment with rapamycin, an mTOR inhibitor, and 2) knockdown of S6 by siRNA transfection. Results: Glutamine stimulated cell proliferation in a dose-dependent manner in the three OC cell lines. In addition, glutamine increased GDH activity, glucose uptake, and lactate and ATP production and resulted in increased phosphorylation of S6 and p42/44. As expected, glutamine starvation had the opposite effect on the OC cell lines, leading to inhibition of cell proliferation, induction of apoptosis and G1 cell cycle arrest, reduced glucose uptake, and decreased lactate and ATP production. Glutamine depletion also induced ROS production and increased expression of the cellular stress proteins PERK, PARP, Bip, and calnexin. Inhibition of S6 by siRNA or rapamycin resulted in inhibition of glutamine-induced cell proliferation as well as a decrease in GDH activity. Conclusions: Glutamine potently increased cell proliferation, glucose uptake, and ATP production whereas glutamine depletion had the opposite effects on OC cells. Glutamine-induced cell proliferation appeared dependent on the mTOR pathway. These studies suggest that targeting glutamine may be a promising therapeutic strategy for ovarian cancer.

S6 inhibition contributes to isoflurane neurotoxicity in the developing brain

Postnatal isoflurane exposure leads to neurodegeneration and deficits of spatial learning and memory in the adulthood. However, the underlying mechanisms remain unclear. Ribosomal protein S6 is demonstrated to play a pivotal role in control of cell survival, protein synthesis and synaptogenesis for brain development. In this study, the possible role of S6 and its upstream signaling pathways in the developmental neurotoxicity of isoflurane was evaluated using models of primary cultured hippocampal neurons and postnatal day 7 rats. We found that isoflurane decreased IGF-1 level and suppressed activation of IGF-1 receptor, sequentially inhibiting S6 activity via IGF-1/MEK/ERK and IGF-1/PI3K/Akt signaling pathways. S6 inhibition enhanced isoflurane-induced decreased Bcl-xL and increased cleaved caspase-3 and Bad, also reduced PSD95 expression and aggravated deficits of spatial learning and memory. S6 activation could reverse the damages above. These results indicate that S6 inhibition, led by suppression of upstream IGF-1/MEK/ERK and IGF-1/PI3K/Akt signaling pathways, is involved in the neuroapoptosis, synaptogenesis impairment and spatial learning and memory decline caused by postnatal isoflurane exposure. S6 activation may exhibit protective potential against developmental neurotoxicity of isoflurane.

The synergistic interaction of MEK and PI3K inhibitors is modulated by mTOR inhibition

Background:Combined targeting of MAPK and PI3K signalling pathways may be necessary for optimal therapeutic activity in cancer. This study evaluated the MEK inhibitors AZD6244 and PD0325901, alone and in combination with the dual mTOR/PI3K inhibitor NVP-BEZ235 or the PI3K inhibitor GDC-0941, in three colorectal cancer cell lines.Methods:Growth inhibition, survival and signal transduction were measured using the Sulforhodamine B assay, clonogenicity and western blotting, respectively, in HCT116, HT29 and DLD1 cell lines.Results:All MEK/PI3K inhibitor combinations exhibited marked synergistic growth inhibition; however, GDC-0941 displayed greater synergy in combination with either MEK inhibitor. NVP-BEZ235 exhibited stronger inhibition of 4EBP1 phosphorylation, and similar inhibition of S6 and AKT phosphorylation, compared with GDC-0941. Both PD0325901 and AZD6244 inhibited ERK phosphorylation, and with MEK/PI3K inhibitor combinations inhibition of S6 phosphorylation was increased. The reduced synergy exhibited by NVP-BEZ235 in combination with MEK inhibitors, compared with GDC-0941, may be due to inhibition of mTOR, and the addition of the mTORC1/2 inhibitor KU0063794 compromised the synergy of GDC-0941:PD0325901 combinations.Conclusion:These studies confirm that dual targeting of PI3K and MEK can induce synergistic growth inhibition; however, the combination of specific PI3K inhibitors, rather than dual mTOR/PI3K inhibitors, with MEK inhibitors results in greater synergy.

Abstract 1668: Pharmacodynamic biomarker development for INK128, a potent and selective inhibitor of TORC1/2 for the treatment of cancer

Abstract Pharmacokinetic / pharmacodynamic (PK/PD) modeling is a valuable strategy to achieve target inhibition as well as predictable and meaningful therapeutic efficacy. The mammalian target of rapamycin (mTOR) comprises two protein complexes, TORC1 and TORC2, which together regulate cell growth, metabolism, angiogenesis, and cell survival. Because TORC1 and TORC2 play a crucial role in several pathways that are frequently dysregulated in human cancer, the mTOR kinase is a compelling target for oncology drug development. Through rational drug design we have identified INK128, a potent and selective small molecule, ATP-competitive, active-site TORC1/2 kinase inhibitor with excellent drug-like properties. Inhibition of phosphorylation of S6 and 4EBP1, downstream markers of TORC1 signaling, was selected for PD analysis in peripheral blood cells (PBCs), skin tissue, and tumor tissue biopsy in mice xenograft tumor models. Time- and dose-dependent inhibition of S6 and 4EBP1 was demonstrated in PBCs by phospho-flow (FACS) analysis. Immunohistochemistry and immunoblot analysis demonstrated a correlation between S6 and 4EBP1 inhibition in tumors or skin tissue and antitumor effect. Additionally, site-selective inhibition of AKT phosphorylation at Ser473, the downstream substrate of TORC2, was also demonstrated in tumors and skin biopsies in mouse xenograft models. Our results demonstrate that daily, oral administration of INK128 selectively inhibits PI3K/AKT/mTOR signaling at the level of TORC1/2, and show that INK128 inhibits growth, and in some cases induces regression, of various tumor xenograft models. Results from these studies display a clear pharmacokinetic and pharmacodynamic relationship. Moreover, the activity of several of these downstream markers can be reproducibly measured in human peripheral blood cells and may permit development of a PK/PD model that might assist to predict PBC and skin tissue PD marker inhibition time-profiles in patients. In summary, INK128 presents a compelling, biomarker-guided approach for the treatment of a variety of cancer by targeting TORC1/2 signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1668.

Abstract 2503: PI3K/Akt/mTOR activity is a major determinant for the sensitivity of NPM-ALK (+) anaplastic large cell lymphoma cells to ALK inhibition

Abstract Anaplastic large cell lymphomas (ALCLs) are a unique subgroup of high grade non-Hodgkin lymphomas. Approximately 60% to 70% of ALCLs contain a t (2:5) (p23; q35) chromosomal rearrangement, generating the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein. NPM-ALK encodes a constitutively activated tyrosine kinase directly involved in the pathogenesis of ALCL. Recent studies have provided solid proof-of-concept validation that inhibition of ALK is sufficient to attenuate the growth and proliferation of ALK (+) ALCL cells. However, differential sensitivity to an ALK inhibitor among human NPM-ALK (+) ALCL cell lines has also been observed. In this study, using 2 fused pyrrolocarbazole (FP)-derived ALK inhibitors and a diaminopyrimidine (DAP)-derived ALK inhibitor as tools, the sensitivity of ALCL cells to ALK inhibition and the signaling pathway(s) that may contribute to the differential sensitivity of ALCL cells to ALK inhibition were characterized and compared. In all four NPM-ALK (+) ALCL cell lines tested, inhibition of Stat3 phosphorylation was consistent with ALK inhibitory activity. In contrast, inhibition of Akt phosphorylation by the FP- or DAP-ALK inhibitor was only observed in the more sensitive cell lines, Sup-M2 and Sudhl-1 cells while in less sensitive cell lines, Karpas-299 and SR-786 cells, Akt was constitutively activated even when ALK phosphorylation was completed abolished. Treatment with LY-294002, a PI3K inhibitor, at 30 µM completely inhibited Akt phosphorylation and led to partial growth inhibition of Karpas-299 and SR-786 cells. Co-treatment with LY-294002 and the FP- or DAP-ALK inhibitor significantly enhanced the cytotoxicity against Karpas-299 and SR-786 cells, similar to the degree observed in Sup-M2 or Sudhl-1 cells treated with the same concentration of ALK inhibitor alone, suggesting PI3K/Akt activity is a major determinant for the sensitivity of NPM-ALK (+) ALCL to ALK inhibition. Karpas-299 subcutaneous tumor xenografts displayed faster growth rate than Sup-M2 tumor xenografts in mice, and the DAP-ALK inhibitor was less efficacious against Karpas-299 tumor xenografts (about 40% TGI and 55% TGI respectively @ 30 or 55 mg/kg, bid, po) compared to that observed against Sup-M2 tumor xenografts (partial and near complete tumor regression respectively at the same dose regimes). Treatment of mice bearing Karpas-299 tumor xenografts with rapamycin, an mTOR inhibitor, @ 10 mg/kg, ip led to the inhibition of S6 and eIF4G phosphorylation in tumor xenografts. Co-administration of rapamycin (10 mg/kg, qd, ip) and the DAP-ALK inhibitor (55 mg/kg, bid, po) significantly enhanced the anti-tumor efficacy against Karpas-299 tumor xenografts and led to near complete tumor regression. These data further support that PI3K/Akt/mTOR activity is a major determinant for the sensitivity of NPM-ALK (+) ALCL to ALK inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2503.

Pharmacodynamic (PD) study of pre-prostatectomy rapamycin in men with advanced localized prostate cancer (PC): A DOD Prostate Cancer Clinical Trials Consortium Trial

5001 Background: Rapamycin is an orally bioavailable and generic mTOR inhibitor with an MTD of 6 mg daily and anticancer activity in solid tumors. We sought to determine the optimal biologic dose (OBD) in the pre-operative setting in men with PC. Methods: We explored the safety and PD activity of 3 and 6 mg of daily oral rapamycin for 14 days prior to radical prostatectomy (RP) in cohorts of 21 men with intermediate risk localized PC. Ten untreated control subjects were included using identical inclusion criteria. Men had Gleason >6 PC involving multiple cores. PD markers in pre-treatment prostate biopsies and RP specimens included p-S6 and p-Akt, 4EBP-1, PTEN, p27, Ki-67, and cleaved caspase-3. Tissue and whole blood sirolimus levels were measured. A Simon 2-stage design using PD efficacy (tumor S6 inhibition > 60%) was utilized. Results: 32 subjects were accrued to this 2 stage pharmacodynamic trial, including 10 control subjects. Median pre-treatment PSA was 6.4 ng/dl, age 60y, Gleason 7 in 85%. 20 accrued to the 3 mg cohort without DLT. However, 2/2 men enrolled at the 6 mg dose level experienced DLT consisting of thrombocytopenia leading to a delay in surgery (<100K) and fever with grade 3 stomatitis. AEs observed in the 3 mg cohort included stomatitis (2), rash (1), post-operative ileus (2), and mild neutropenia (2). One post-operative ileus was observed among control patients. PD studies demonstrated tumor S6 inhibition in >50% of subjects (median 60% decline, p=0.026 vs. baseline) with no negative effect on the state of Akt phosphorylation (p=0.82) or p27 levels (p=0.10). Prostate sirolimus levels (range 7.1–47.2 ng/g) were two-fold higher than whole blood concentrations (range 3.2–19.2 ng/ml). There was no effect of rapamycin on rates of post-operative wound healing or bleeding. PBMC S6 activity inhibition did not correlate with tumor S6 inhibition. Conclusions: The MTD/OBD of daily rapamycin in the pre-operative or prevention setting is 3 mg. This dose demonstrated downstream mTOR inhibition in tumor tissue and achieved adequate prostate tissue levels. Additional PD and genomic studies will be presented. Conducted through the DOD PCCTC and registered at clinicaltrials.gov as NCT00311623 . [Table: see text]