High Levels Of pAKT Research Articles

Acquired in Vitro Resistance to Ibrutinib Is Associated with Transcriptional Re-Programming and Sustained Survival Signaling in Waldenströms Macroglobulinemia and Mantle Cell Lymphoma, Independent of BTK Cys481 Mutation

Background: While B-cell receptor (BCR) signaling is essential for the development, of normal B cells, its aberrant hyper-activation results in neoplastic transformation of B-lymphocytes. Recent investigations using small molecule inhibitors validate the BCR pathway as a valuable target. Bruton’s tyrosine kinase (BTK) is one of the components of a signaling hub that transduces signals from the BCR into the cell for its activation and has been shown to be a therapeutic target. Ibrutinib (PCI-32765), an irreversible BTK inhibitor has shown clinical efficacy in CLL, mantle cell lymphoma (MCL) and Waldenströms macroglobulinemia (WM). Ibrutinib binds to cysteine-481 of the BTK protein and blocks its phosphorylation, resulting in termination of BCR-mediated activation of cells with a concomitant induction of death. Despite the clinical success of ibrutinib, a high percentage of patients achieve only partial response and eventually acquire resistance to the drug, resulting in aggressive relapse of the disease. A mutation of Cys481-Ser in BTK (ibrutinib-BTK binding site) has been reported to be one of the reasons for the development of ibrutinib resistance (IR). To understand the mechanisms resulting in acquisition of IR, we developed preclinical models of IR in WM and MCL.Materials: Ibrutinib was obtained from Pharmacyclics, CA. Validated human WM models (BCWM.1, RPCI-WM1 and MWCL.1 cell lines) and human MCL models (Jeko-1 and Maver cell lines) were used for the study.Results: BTK was constitutively phosphorylated at Y223 and Y551 in all the cell lines tested and this was inhibited by ibrutinib in a dose dependent manner. Phosphorylation of other kinases in the cascade such as SYK (Y323 and Y525/526) and PLCg2 (Y759 and Y1217) were also inhibited while AKT phosphorylation at both Ser473 and Thr308 was consistently increased in presence of ibrutinib. Treatment with ibrutinib induced cell cycle arrest in the G1 phase by 24h followed by apoptosis. Cell growth assays (MTS assay) showed that BCWM.1 was the most sensitive cell line followed by MWCL-1, RPCI-WM1, Maver and Jeko-1. Exposure of WM and MCL cells for prolonged periods of time with progressively increasing concentrations of ibrutinib resulted in outgrowth of clones (IR WM and MCL cell lines) that were resistant to apoptosis with a slow growth rate as compared to wild type parental cells. IR cells attained 2 – 20 fold resistance to ibrutinib as compared to the respective parental lines as determined by MTS assay. Sequence analysis of the BTK gene in all the cell lines revealed no mutation in IR cells at Cys481 suggesting that in an acquired IR state, resistance to ibrutinib can be developed independent of BTK Cys481 mutation. Interestingly, we found p-BTK levels to be markedly reduced in IR cells. Ibrutinib reversal experiments suggested that while a continuous presence of ibrutinib is needed for inhibition of BTK phosphorylation, a stable IR state could be maintained (for >1 month) in the absence of ibrutinib. This suggested the cells reliance on a parallel survival pathway, independent of BTK phosphorylation. Focused mRNA (Nanostring nCounter assay) and immunoblot analysis revealed significant changes in the expression profiles of several cellular elements. These included transcription factors such as PU.1, IRF4, BLIMP1, BCL-6 b-catenin as well as the phosphorylated ERK1/2, STAT1 and 3 suggesting a reprogramming of critical cellular networks, which IR tumor cells might be utilizing to overcome ibrutinib-induced cytotoxicity. Importantly, we observed that IR cells retained high levels of p-AKT and showed an increase in expression of BCL2 family members, as well as BCL-2 itself. Treatment of IR cells with ibrutinib +/- MK2206 (AKT inhibitor), or ABT-199 (BCL-2 inhibitor), synergistically induced cytotoxicity in IR cells, suggesting the importance of these parallel survival pathways (AKT/BCL2) in maintaining an IR state.Conclusion: Here we demonstrate that in the absence of BTK Cys481 mutation, an IR state is associated with reprogramming of transcriptional networks countering ibrutinib-induced toxicity by activation of AKT and BCL-2. Our current data exposes multiple vulnerabilities within IR cells, which can be therapeutically exploited to potentially delay onset of IR, by targeting alternative oncogenic mechanisms that are activated in presence of sustained BTK inhibition. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4513: IMGN289, an EGFR-targeting antibody-drug conjugate, is effective against tumor cells that are resistant to EGFR tyrosine kinase inhibitors

Abstract EGFR is one of the oncogenic drivers in non-small-cell lung adenocarcinoma (NSCLC AC). EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib, gefitinib and afatinib, show remarkable single agent activity in NSCLC AC with EGFR mutations. However, a majority of the tumors develop resistance within twelve months. T790M secondary EGFR mutation and activation of alternative signaling pathways, such as MET, are the two most common resistance mechanisms. To address this clinical challenge, we developed IMGN289, an EGFR antibody-drug conjugate consisting of the novel J2898A antibody (Ab), which selectively binds to and inhibits EGFR-driven tumor cell growth, conjugated to the maytansinoid DM1, a potent anti-microtubule agent, via the SMCC thioether linker. IMGN289 showed significantly enhanced cytotoxic activity, relative to cetuximab or unconjugated J2898A, against tumor cell line models that are dependent on EGFR signaling (Chittenden et al., AACR 2013). To evaluate the ability of IMGN289 to overcome mechanisms of EGFR TKI resistance, IMGN289 activity was tested in erlotinib-resistant EGFR-mutant cell lines which overexpressed MET (HCC827-ER) or carry the T790M EGFR mutation (HCC827-EPR) (Suda et al., Clin Cancer Res 2010). Despite variable levels of EGFR expression in these cell lines, (193,000, 32,500 and 422,000 antigens per cell in HCC827, HCC827-ER and HCC827-EPR, respectively), IMGN289 showed comparable cytotoxic activities against these cell lines in vitro. In addition to MET overexpression, the MET pathway can also be activated by HGF, the MET ligand. While addition of HGF made the HCC827 cell line resistant to EGFR TKIs (Yano et al., Cancer Res 2008) and decreased growth inhibition by cetuximab, IMGN289 activity was unaffected. Additionally, we generated an erlotinib-resistant HCC827-ER-E4 cell line by exposing the EGFR-mutant HCC827 cells to increasing concentrations of erlotinib for ∼6 months, followed by subcloning. The HCC827-ER-E4 cell line expressed mesenchymal markers and acquired a mesenchymal phenotype. It expressed lower levels of EGFR, HER3 and MET compared to the parental cell line and showed no loss of PTEN. In the presence of erlotinib, HCC827-ER-E4 cells retained high levels of pAKT and pERK, suggesting that activation of alternative signaling pathways may be responsible for the erlotinib resistance. In vitro cytotoxic assays showed that IMGN289 was equally cytotoxic to the parental and the HCC827-ER-E4 cell line. In conclusion, IMGN289 demonstrated EGFR-targeted cytotoxicity against NSCLC cell lines that harbor some of the most common mechanisms of resistance to EGFR TKIs. IMGN289 represents a promising novel candidate for treatment of EGFR-expressing NSCLC AC that has acquired resistance to EGFR TKIs. Citation Format: Yulius Y. Setiady, Ling Dong, Anna Skaletskaya, Jan Pinkas, Robert J. Lutz, John M. Lambert, Thomas Chittenden. IMGN289, an EGFR-targeting antibody-drug conjugate, is effective against tumor cells that are resistant to EGFR tyrosine kinase inhibitors. [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 4513. doi:10.1158/1538-7445.AM2014-4513

Abstract 4517: Evaluation of the TOR kinase inhibitor CC214-2 in a mouse model of hormone-refractory prostate cancer

Abstract Prostate cancer is one of the most common malignancy diagnosed in men. Although androgen deprivation therapy is effective at the beginning of treatment, the majority of patients eventually progress to hormone refractory prostate cancer (HRPC), which leads to a poor outcome. HRPC mouse models have been developed to study transition from hormone dependent (HD) to hormone refractory (HR) disease; however, the exact mechanism behind this phenotypic conversion remains unclear. By using serial in vivo transplantation of LNCap xenograft tumors in castrated male SCID mice, we developed, validated and characterized a mouse model of HRPC, HR-LNCap. In vivo passaged tumor fragments form solid tumors in castrated male SCID mice. By comparison, HR-LNCap tumors grow more aggressively than parental LNCap tumors in SCID mice. A tumor cell line established from HR-LNCap xenograft tumors was able to grow in charcoal stripped serum containing medium, indicating it is androgen independent. HR-LNCap tumors lost sensitivity to androgen receptor antagonist bicalutamide, but remained sensitive to MDV-3100, a second generation AR antagonist approved for treating HRPC. Characterization of this model revealed that HR-LNCap tumors had unique AR signaling pathway profiles, including elevated AR expression and decreased expression of the AR-regulated genes, prostate specific antigen (PSA) and FKBP5, when compared with parental LNCap tumors. RNA sequencing was performed on HR-LNCap tumors to determine expression signature of the model. Interestingly, additional pathway analysis showed that HR-LNCap tumors have significantly higher levels of pAKT in comparison with parental LNCap tumors, indicating activation of AKT/mTOR pathway. Treatment of HR-LNCap tumor bearing mice with a small molecule TOR kinase inhibitor CC214-2 showed significant tumor growth inhibition. Further analysis demonstrated that treatment with CC214-2 inhibited TORC1 and TORC2 pathway markers, pAKT, pS6 and p4EBP1, in HR-LNCap tumors. Taken together, this study describes a mouse model of HRPC with a unique molecular profile, which can be used to evaluate novel therapeutics against HRPC. Citation Format: Wenqing Yang, Jing Jiu, Rama Krishna Narla, Heather K. Raymon. Evaluation of the TOR kinase inhibitor CC214-2 in a mouse model of hormone-refractory prostate cancer. [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 4517. doi:10.1158/1538-7445.AM2014-4517

Abstract P5-09-07: Identification of resistance-specific gene expression signatures in a breast cancer patient-derived xenograft with acquired resistance to different endocrine therapies

Abstract Background: Acquired resistance to endocrine treatments (ET) occurs in more than 70% of cases of luminal breast cancer (LBC). We used patient derived xenografts (PDX) models of LBC to study molecular changes associated with acquired resistance to different ET modalities. Methods: A PDX model of LBC, established from an early stage BRCA2-mutated breast cancer, was treated with different ET (tamoxifen, fulvestrant, oophorectomy and letrozole) during several months. Tumors escaping to therapies were re-engrafted and maintained under therapy. ET-resistant and parental hormono-responders tumors were analyzed with immunohistochemistry (IHC), RT-PCR and Affymetrix Gene Expression Arrays. Hormono-resistant tumors were additionally studied for their in vivo response to ET, mTOR and PARP inhibitors. Results: From the initially ET sensitive HBCx22 xenograft model (Cottu, BCRT 2012) two resistant models were obtained respectively to tamoxifen (HBCx22-TamR) and to estrogen deprivation (HBCx22-OvaR). Unsupervised clustering of gene expression showed a clear cut separation between parental, TamR and OvaR tumors. Genes differentially expressed in TamR and OvaR tumors compared to parental HBCx22 were only partially overlapping. Up-Regulated genes in both TamR and OvaR tumors (n = 302) were involved in response to wounding, nucleotide metabolism, immune system, adhesion and cell growth. Biological Processes (BP) specifically deregulated in OvaR tumors (n = 380) included embryonic development, antigen presentation, amino acid and lipid metabolism. The top BP specifically regulated in TamR tumors (n = 1059) were response to estrogen and steroid hormones, TGF-b signaling, hypoxia, regulation of cell proliferation, with several strongly up-regulated genes of the histone clusters 1 and 3. Ingenuity Transcription Factor Analysis predicted activation of NFKB, SP1, AP-1 and JUN, and inhibition of ESR1. RT-PCR and IHC analyses confirmed the down regulation of ER controlled genes in the TamR tumors. Expression of ER co-regulators determined by RT-PCR showed that GREB1 was strongly reduced in TamR, while PBX1, GATA3 and FOXA1 were inhibited in OvaR. IHC analysis showed a loss of PTEN expression in HBCx22, with high levels of p-AKT and p-RPS6 in both parental and TamR and OvaR tumors. In vivo ET showed that the TamR xenograft was resistant to all modalities of ET, while OvaR was resistant to estrogen deprivation while retaining some sensitivity to tamoxifen and fulvestrant. Treatment with the mTOR inhibitor RAD001 arrested tumor growth but did not show any additive effect when combined to ET in TamR or OvaR tumors. Conversely, the combination of RAD001 with Olaparib was highly synergistic and induced complete tumor response in 70% of mice. Conclusions: According to the therapeutic selection, tumors derived from a PDX model of ER+ breast cancer show specific resistance patterns and gene expression profiles including disruption in the ER transcriptional program. The analysis of additional resistant tumors established from a second ER+ PDX will be presented at the meeting. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-07.

ROR1 Can Interact With TCL1 and Enhance Leukemogenesis In Eµ-TCL1 Transgenic Mice

ROR1 is an onco-embryonic antigen found on chronic lymphocytic leukemia (CLL) B cells, but not on normal adult tissues. We generated B6 transgenic (Tg) mice with human ROR1 regulated by the murine Ig promoter/enhancer (ROR1 Tg). Such animals had B-cell-restricted expression of ROR1 and infrequently developed ROR1+/CD5+/B220low leukemia resembling human CLL at ≥15 months of age. The leukemia cells of these animals had phenotypic features in common with those of the leukemia that originates in B6 Eµ-TCL1-Tg (TCL1) mice, which develop a CLL-like disease at ≥ 9 months of age. However, in contrast to human CLL, the leukemia that develops in TCL1 Tg mice does not express ROR1, indicating that expression of this antigen is not necessary for leukemogenesis. However, in immune-precipitation and mass spectrometry studies we found that ROR1 could complex with TCL1 in human CLL cells. TCL1 is a proto-oncogene product that can serve as a co-activator of AKT. To investigate whether expression of ROR1 could affect the biology of the leukemia that develops in TCL1 Tg mice, we crossed our ROR1 Tg animals with TCL1-Tg mice. Progeny with both transgenes (ROR1xTCL1) developed CD5+/B220low B-cell leukemia at a significantly younger median age than did littermates with either transgene alone. ROR1xTCL1 leukemia B-cells had higher levels of pAKT than TCL1 leukemia-cells and expressed high-levels of human ROR1, which we also found could complex with TCL1. Exploratory subnetwork analyses of transcriptome microarray data on isolated leukemia cells using Ingenuity Pathway network tools revealed 51 subnetworks that were expressed at different levels between the two types of leukemia, 21 of which had z scores in excess of 0.8, and an associated functional annotation with a false-discovery-rate (FDR) of less than 0.05. This analysis implied that ROR1xTCL1 leukemia cells had higher expression of subnetworks implicated in embryonic and tumor-cell proliferation, but lower expression of subnetworks involved in cell-cell adhesion or cell-death, than did TCL1 leukemia-cells. ROR1xTCL1 leukemia-cells also had higher proportions of Ki-67-positive cells, lower proportions of cells undergoing spontaneous apoptosis, and produced more aggressive disease upon adoptive transfer than TCL1 leukemia-cells.We examined the activity of two different mouse anti-human ROR1 mAbs, D10 and 4A5, which bind to distinct non-overlapping epitopes of ROR1, as assessed in cross-blocking studies. Treatment of ROR1xTCL1 leukemia cells with D10 in vitro resulted in reduced expression of pAKT within 1 hour after addition of the antibody to the leukemia cells, an effect that was not apparent in control Ig or 4A5-treated cells. We treated ROR1 Tg mice engrafted with CD5+/B220low/ROR1+ leukemia cells with intravenous injections of D10, 4A5, or control mouse IgG (mIgG), at 10 mg/kg. At five weeks, mice given D10 in one representative experiment had significantly fewer CD5+/B220low leukemia cells (2.4 ± 1.0 x106, n=3) in the blood than mice that received mIgG (2.0 ± 0.3 x107, n=3, p=0.032), whereas the number of leukemia cells in the blood of mice given 4A5 (1.6 ± 0.3 x107, n=3, p>0.05) was not significantly different than that of mice treated with mIgG. Furthermore, mice that received CD5+/B220low/ROR1+ B cells and that were treated with D10 had significantly smaller spleens than mice that were treated with mIgG or 4A5. Although D10 was effective in inhibiting the engraftment of ROR1xTCL1 leukemia cells, administration of either anti-ROR1 mAb had no effect on the endogenous non-leukemia (CD5-/B220Hi/ROR1+) B cells or T cells, or on engraftment of leukemia cells from TCL1 Tg mice, which do not express ROR1. Our data demonstrate that ROR1 accelerates progression of TCL1 leukemia and may be a target for therapy of patients with CLL. Disclosures:No relevant conflicts of interest to declare.

Transcription Activator-Like Effector Nucleases (TALENs)-Mediated Deletion Of MIR17HG In Burkitt Lymphoma Cells Decreases mTOR Pathway Activity and Increases Chemosensitivity

BackgroundBurkitt lymphoma (BL) is the most common form of non-Hodgkin lymphoma in children. Although curable in most cases, those patients who relapse have a dismal prognosis with a 4-year survival of 16% (Cairo, Blood 2007). Identification of biomarkers to predict relapse/refractory disease is needed, and an understanding of the underlying mechanisms is vital to develop novel targeted therapies. We previously identified a recurrent gain of chromosome 13q31 that was associated with relapse and increased expression of miR-17-92 in pediatric BL (Schiffman/Miles, Br J Haematol, 2011). In the present study, we sought to determine the role of miR-17-92 expression in vitro using TALENs mediated deletion of the miR-17-92 locus (MIR17HG) in Raji BL cells that express high levels of miR-17-92 (Shiffman/Miles, 2011). MethodsThe modified restriction enzyme and ligation assembly method (Lee/Cairo, ASH 2012) was used to construct MIR17HG TALENs. Expression of miR-17 was assayed by TaqMan assay, and expression of the miR-17-92 target PTEN mRNA and protein were assayed by RT-PCR and western blot, respectively. Proliferation and caspase activity were measured at baseline and after treatment with cyclophosphamide. AKT and phospho (p)AKT were assayed by western blotting. To evaluate mTOR and MAPK pathway activation, pS6 and pERK were assayed by phosphoflow cytometry. In addition, caspase 3 activity was assayed by phosphoflow cytometry to assess induction of apoptosis. Cells were treated with chemotherapy (cyclophosphamide or 4-hydroperoxycyclophosphamide, 4HC), rapamycin, the PI3 kinase inhibitor PIK90, and the dual PI3K/mTOR inhibitor BEZ235 (Axon Medchem). To further validate the role of miR-17 in chemoresistance, miR-17 expression levels were measured by RT-PCR TaqMan assay in the previously developed rituximab-chemotherapy resistant Raji cell lines (RCRCL) Raji 2R and Raji 4RH (Barth et al. Br J Haematol, 2012). ResultsMIR17HG TALENs were used to generate two Raji cell lines with a hemizygous deletion of MIR17HG, #31(+/-) and #48(+/-). Relative to vector control Raji cells, cell lines with hemizygous MIR17HG deletion showed lower expression of miR-17: Raji = 1.0, #31= 0.80-fold, and #48= 0.71-fold. PTEN mRNA was higher in #31 and #48 cells than in the control (Raji = 1.0, #31 = 1.38-fold, and #48 = 2.24-fold), but there was no apparent difference in PTEN protein by western blot. Western blotting showed decreased pAKT with no difference in total AKT. Cyclophosphamide (10mM) induced a significant decrease in proliferation at 48 hours (90.5 +/- 2.9%, p=0.007) in #31 cells vs. control cells that was associated with a trend toward increased caspase 3/7 activity (148.3+/-8.9%, p=0.08). By phosphoflow cytometry, Raji #48 cells showed decreased pS6 compared to control cells and a modestly greater induction of caspase 3 in response to 4HC (3.4 uM) or PIK90 after 48 hours. The combination of 4HC and PIK90, however, significantly induced caspase 3 in Raji #48 cells relative to control Raji cells. Raji cells showed high basal levels of pERK and pS6. BEZ235 or PIK90 treatment decreased p-ERK. Rapamycin did not affect p-ERK but did decrease pS6. While BEZ235 and 4HC each modestly induced caspase alone in Raji cells, the combination caused a much greater increase in caspase 3. In RCRCLs Raji 2R and Raji 4RH, which show high pAKT levels by western blot and phosphoflow cytometry, miR-17 levels were increased (1.73- and 1.69-fold, respectively) relative to chemosensitive Raji cells (1.0) that exhibit lower pAKT levels. ConclusionsIncreased miR-17-92 expression in BL may contribute to therapy resistance, which is further supported by finding increased miR-17 expression in chemotherapy resistant Raji cell lines. Hemizygous deletion of the miR-17-92 locus in BL cells led to increased PTEN mRNA, decreased mTOR pathway activation (decreased pAKT and pS6), and increased sensitivity to chemotherapy. Resistance to PIK90 and 4HC in parental Raji cells that overexpresses miR-17-92 may be due in part to a compensatory upregulation of the MAPK and mTOR pathways, as demonstrated by increased pERK and pS6. Inhibiting this survival mechanism with the PI3K/mTOR inhibitor BEZ235 restored sensitivity to cytotoxic chemotherapy. These findings suggest potential mechanisms underlying BL therapy resistance as well as targeted therapies to overcome resistance. Disclosures:Cairo:Roche/Genentech: advisory board Other.

Activation of the PI3K/AKT/mTOR pathway in diffuse large B cell lymphoma: clinical significance and inhibitory effect of rituximab

Diffuse large B cell lymphoma (DLBCL) represents the most common subtype of non-Hodgkin lymphoma and accounts for approximately 30% of newly diagnosed lymphoid neoplasms in Western countries, and 40-50% in China. A better understanding of the biology of DLBCL is needed for the development of potential therapeutic agents that target specific intracellular pathways. In this study, expression of the important components of the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway and their clinical significance were investigated in 73 DLBCL cases. The effect of rituximab alone or combined with the PI3K/AKT/mTOR pathway inhibitor rapamycin was further evaluated in the DLBCL cell lines. A total of 73 patients were identified, including 45 men and 28 women aged 18 to 78 years (median age 50 years). Of these patients, p-AKT was positive in 40 cases (54.8%), p-p70S6K in 34 cases (46.6%), and p-4E-BP1 in 33 cases (45.2%). Activation of the PI3K/AKT/mTOR pathway was related to poor disease outcome in DLBCL patients treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) but not in those treated with rituximab-CHOP. Rituximab combined with rapamycin synergically downregulated the PI3K/AKT/mTOR signaling pathway. Western blot analysis revealed a baseline activation status of the PI3K/AKT/mTOR pathway in DLBCL cell lines, with high levels of p-AKT, p-mTOR, in addition to downstream molecules p-p70S6K and p-4E-BP1. The results indicate that the PI3K/AKT/mTOR pathway is a potentially important signaling route and an unfavorable prognostic factor for DLBCL. Patients with PI3K/AKT/mTOR activation experience a more rapidly deteriorating clinical course with poor treatment response and decreased survival time. Addition of rituximab could downregulate PI3K/AKT/mTOR activation, reversing its negative effect on chemotherapy-treated patients. In addition, our results indicate that the combination of rituximab and inhibition of the activated PI3K/AKT/mTOR pathway could be a promising target for DLBCL therapeutic intervention in the future.

G-CSF activation of AKT is not sufficient to prolong neutrophil survival

Neutrophils play an important role in the innate immune response against bacterial and fungal infections. They have a short lifespan in circulation, and their survival can be modulated by several cytokines, including G-CSF. Previous studies have implicated AKT as a critical signaling intermediary in the regulation of neutrophil survival. Our results demonstrate that G-CSF activation of AKT is not sufficient to prolong neutrophil survival. Neutrophils treated with G-CSF undergo apoptosis, even in the presence of high levels of p-AKT. In addition, inhibitors of AKT and downstream targets failed to alter neutrophil survival. In contrast, neutrophil precursors appear to be dependent on AKT signaling pathways for survival, whereas high levels of p-AKT inhibit proliferation. Our data suggest that the AKT/mTOR pathway, although important in G-CSF-driven myeloid differentiation, proliferation, and survival of early hematopoietic progenitors, is less essential in G-CSF suppression of neutrophil apoptosis. Whereas basal AKT levels may be required for the brief life of neutrophils, further p-AKT expression is not able to extend the neutrophil lifespan in the presence of G-CSF.

IS1-4 - Synergistic Anti-Tumor Effect of Sorafenib in Combination with AKT Inhibitors in Hepatocellular Carcinoma

ABSTRACT Background Sorafenib is currently the only approved agent for hepatocellular carcinoma (HCC). However, its clinical efficacy in HCC is limited. Previous studies indicate that compensatory activation of AKT may contribute to the resistance to sorafenib. We hypothesized that combination of sorafenib and AKT inhibitors could enhance the antitumor effect of sorafenib in HCC. Methods We analyzed the cellular and molecular effects of sorafenib in combination with inhibitors of AKT in human HCC cell lines, and evaluated their combinational effects against HCC in vitro and in vivo. Results HCC cells, including Huh7, Hep3B, and SKhep1 cells, expressed high levels of p-AKT in a prompt and sustained manner upon treated with sorafenib. Down-regulation of AKT by siRNA or inhibition of phospho-AKT by AKT inhibitors (MK-2206 or perifosine) enhanced the anti-proliferative effect of sorafenib in cultured HCC cells. The combination indexes of sorafenib and AKT inhibitors were Conclusions Inhibition of AKT by AKT inhibitors improved the antitumor effect of sorafenib against HCC in vitro and in vivo. Combination of AKT inhibitors and sorafenib warrants further consideration for clinical development in HCC. (The study was supported by NSC97-2628-B-002-004-MY3 and NSC100-2325-B-002-043-.)

P53 Stabilization Induces Cell Growth Inhibition and Affects IGF2 Pathway in Response to Radiotherapy in Adrenocortical Cancer Cells

Adrenocortical carcinoma (ACC) is a very rare endocrine tumour, with variable prognosis, depending on tumour stage and time of diagnosis. However, it is generally fatal, with an overall survival of 5 years from detection. Radiotherapy usefulness for ACC treatment has been widely debated and seems to be dependent on molecular alterations, which in turn lead to increased radio-resistance. Many studies have shown that p53 loss is an important risk factor for malignant adrenocortical tumour onset and it has been reported that somatic mutations in TP53 gene occur in 27 to 70% of adult sporadic ACCs. In this study, we investigated the role of somatic mutations of the TP53 gene in response to ionizing radiation (IR). We studied the status of p53 in two adrenocortical cell lines, H295R and SW-13, harbouring non-functioning forms of this protein, owing to the lack of exons 8 and 9 and a point mutation in exon 6, respectively. Moreover, these cell lines show high levels of p-Akt and IGF2, especially H295R. We noticed that restoration of p53 activity led to inhibition of growth after transient transfection of cells with wild type p53. Evaluation of their response to IR in terms of cell proliferation and viability was determined by means of cell count and TUNEL assay.wtp53 over-expression also increased cell death by apoptosis following radiation in both cell lines. Moreover, RT-PCR and Western blotting analysis of some p53 target genes, such as BCL2, IGF2 and Akt demonstrated that p53 activation following IR led to a decrease in IGF2 expression. This was associated with a reduction in the active form of Akt. Taken together, these results highlight the role of p53 in response to radiation of ACC cell lines, suggesting its importance as a predictive factor for radiotherapy in malignant adrenocortical tumours cases.

Modeling human osteosarcoma in mice through 3AB‐OS cancer stem cell xenografts

Osteosarcoma is the second leading cause of cancer-related death for children and young adults. In this study, we have subcutaneously injected-with and without matrigel-athymic mice (Fox1nu/nu) with human osteosarcoma 3AB-OS pluripotent cancer stem cells (CSCs), which we previously isolated from human osteosarcoma MG63 cells. Engrafted 3AB-OS cells were highly tumorigenic and matrigel greatly accelerated both tumor engraftment and growth rate. 3AB-OS CSC xenografts lacked crucial regulators of beta-catenin levels (E-cadherin, APC, and GSK-3beta), and crucial factors to restrain proliferation, resulting therefore in a strong proliferation potential. During the first weeks of engraftment 3AB-OS-derived tumors expressed high levels of pAKT, beta1-integrin and pFAK, nuclear beta-catenin, c-Myc, cyclin D2, along with high levels of hyperphosphorylated-inactive pRb and anti-apoptotic proteins such as Bcl-2 and XIAP, and matrigel increased the expression of proliferative markers. Thereafter 3AB-OS tumor xenografts obtained with matrigel co-injection showed decreased proliferative potential and AKT levels, and undetectable hyperphosphorylated pRb, whereas beta1-integrin and pFAK levels still increased. Engrafted tumor cells also showed multilineage commitment with matrigel particularly favoring the mesenchymal lineage. Concomitantly, many blood vessels and muscle fibers appeared in the tumor mass. Our findings suggest that matrigel might regulate 3AB-OS cell behavior providing adequate cues for transducing proliferation and differentiation signals triggered by pAKT, beta1-integrin, and pFAK and addressed by pRb protein. Our results provide for the first time a mouse model that recapitulates in vivo crucial features of human osteosarcoma CSCs that could be used to test and predict the efficacy in vivo of novel therapeutic treatments.

Abstract 2279: Akt inhibitor SC-66 leads to inhibition of mTOR pathway in cervical cancer cell line C33A.

Abstract Background We observed previously that overexpression of activated Akt is associated with poor prognosis in cervical cancer patients treated with concurrent cisplatin and pelvic irradiation (communicated). Several inhibitors of the PI3K/Akt/mTOR pathway have been developed, and many are being tested in clinical trials. The purpose of this study was to test the allosteric Akt inhibitor, SC-66 in cervical cancer cell lines and to determine drug sensitivity and inhibition of downstream pathway components. Experimental Design Eight cervical cancer cell lines (CaSki, C33A, C41, HeLa, HT-3, ME-180, SiHa and SW756) were assessed for activation status of Akt (Ser473, Thr308 and Thr450), downstream targets of Akt (FOXO-1, GSK3-α, PRAS40) and mTOR (mTOR, ribosomal S6 kinases (p70S6K), (eukaryotic initiation factor 4E)-binding protein, 4E-BP1 and S6) pathway by western blot. Among these cell lines tested, one with high Akt (C33A) was chosen for further experiments. Cell lines were treated with the allosteric Akt inhibitor SC-66, which interferes with PH domain binding to PIP3 leading to Akt ubiquitination. The dose and time course analysis were done by determining the cell viability using Alamar Blue and EC50 using PRIZM software. Akt and mTOR pathway inhibition were assessed by analyzing phosphorylation of various molecules of the pathway with or without SC-66 (6-10µg/ml) by western blot. Results Expression of activated Akt (S473, T450 and T308) was prominent in C33A. Since C33A expressed high pAkt levels we checked for alterations in the upstream regulators PIK3CA and PTEN (Phosphatase and tensin homolog). C33A harboured G263A (R88Q) alteration in exon1 of PIK3CA and PTEN protein expression was undetected. C33A was found to be the most sensitive cell line towards SC-66 treatment, with an EC50 value of 0.486µg/ml followed by ME-180 (0.82µg/ml), SW756 (1µg/ml) and SiHa (2.5µg/ml). To test the effects of SC-66 on the Akt/mTOR pathway, C33A cells were treated with doses from 6-10µg/ml of SC-66 for 3h and all the mTOR pathway targets were inhibited effectively at 7µg/ml, and Akt substrates between 7-8µg/ml. Akt activates mTOR which in turn phosphorylates p70S6K and 4E-BP1 ultimately resulting in cell growth and proliferation. SC-66 is effective in blocking essentially all these steps of signaling in C33A, a cell line with high activated Akt. Conclusions SC-66 potently inhibits proliferation of cervical cancer cell lines. C33A, a cervical cancer cell line with high levels of pAkt, is highly sensitive to SC-66, resulted in inhibition of phosphorylation of mTOR, 4EBP1 and p70S6K. Since high levels of pAkt are associated with poor outcome after concurrent chemo-radiation in cervical cancer, SC-66 could prove to be an effective therapeutic adjunct in these patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2279. doi:1538-7445.AM2012-2279

Abstract 111: PIK3R1 gene alterations, insights on its role in lung cancer

Abstract Gene abnormalities are the cause of cancer and efforts are still needed to identify all the genes that promote carcinogenesis, including those altered at low frequencies. The occurrence of homozygous deletions (HD) is one mechanism for tumor suppressor gene inactivation. We scrutinized the SNP arrays at the Sanger Database to search for HD in lung cancer cell lines. Several candidate tumor suppressors, including PIK3R1, were found to be altered by HD. PIK3R1 codifies for p85α, a regulatory subunit of the PI3K, and mutations have been found in different tumor types. The PI3K pathway is highly relevant in cancer since it contains well known oncogens and tumor suppressors. Despite that, the functional significance of PIK3R1 mutations is uncertain, because activating and inactivating alterations have been described in tumors. Here, we aimed to clarify the role of this gene in cancer. We amplified by PCR each individual exon of the gene and confirmed the presence of a HD at the NCI-H2171 cell line. Mutational analysis of PIK3R1 was then extended to a panel of 86 lung cancer cell lines. We identified several changes including an heterozygous nonsense mutation, c.211G>T (p.G71*), an heterozygous missense mutation, c.211G>T (p.D13H) and several intronic changes outside the canonical splice sites. Intriguingly, the levels of p85α protein in the cell line carrying the p.G71* mutation were almost zero, indicating the presence of a second hit. To search for the presence of intragenic small deletions, undetectable by single exon amplification, we studied the cDNA of PIK3R1. No additional alterations were detected in this cell line nor in the other cells carrying PIK3R1 heterozygous missense mutations. Next, we used cancer cell lines that have no p85α protein due to HD to determine whether the lack of p85α impinges the AKT-mTOR pathway. To this end, we tested the levels of pAKT-T308, pAKT-S473 and pS6-S235/236, upon different cell culture conditions (i.e. +/−IGF) in cancer cells carrying alterations at distinct components of the PI3K pathway. We found that cells with alterations at ERBB2 and PTEN had constitutive high levels of pAKT and pS6, in all the conditions tested. Surprisingly, some cells lacking p85α protein due to HD at PIK3R1, readily regulated the activation/inactivation of the pathway, accordingly to the cell culture conditions. This indicates that the inactivation of the p85α protein in cancer does not compromise the appropriate modulation of AKT/mTOR signalling, possibly due to a compensation effect by other regulatory subunits. In conclusion, PIK3R1 is mutated in lung cancer, albeit at low frequencies. Furthermore, PIK3R1 alterations contribute to oncogenesis by mechanisms that require further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 111. doi:1538-7445.AM2012-111

Abstract 3812: Deguelin as a novel therapeutic agent for Herceptin resistant breast cancers

Abstract Human breast cancers overexpressing HER-2 are generally highly aggressive. Current therapeutic strategies to target HER-2 signaling pathway with antibodies or small molecule tyrosine kinase inhibitors have shown impressive success in the management of these tumor types, although most women exhibit short term response to these therapies. Acquired resistance to HER-2 target drugs, especially Herceptin is a major challenge to clinicians. Patients receiving Herceptin therapy invariably recur and most often with highly aggressive tumors. Thus identifying new drugs which target multiple molecular pathways is desired to treat Herceptin resistant breast cancer. Using paired HER-2 overexpressing parental Herceptin responsive (BT-474) and its Herceptin resistant variant cells (BT-474HR) we showed that acquired resistance to Herceptin is associated with enhanced expression of HER-2, EGFR, CyclinD1, P-AKT (Ser-473), PI-3K, JAB1 proteins and decreased expression of nuclear pten and p27 proteins. These results are similar to those observed in recurrent breast tumors in patients following Herceptin treatment. Deguelin, a natural product derivative of rotenoids and has been shown to inhibit growth of diverse cancer types. Deguelin is a known AKT inhibitor, however its efficacy is never tested in breast cancers resistant to Herceptin which express high levels of p-AKT. We examined effect of Deguelin on HER-2 overexpressing BT-474 BT-474-HRcells. Deguelin inhibited growth of both, BT-474 and BT-474HR cells in concentration (0-250nM) and time dependent (24-72h) manner. There was a 40-50%- inhibition of both BT474and BT-474HR cells at 25nM Deguelin treatment at 72h. Deguelin decreased cell surface expression of HER-2 in both these cell lines. In parental BT-474 cells both Herceptin 5-10ug/ml and Deguelin down regulated HER-2 expression, but only Deguelin reduced HER-2 (125-250nM) over expression in both BT-474 and BT-474HR cells. Down regulation of HER-2 following Deguelin treatment was associated with simultaneous down regulation of nuclear accumulation of cyclin D1, and JAB1, and p-AKT (Ser-473) proteins and up regulation of nuclear p27 and p53in both BT-474 and BT474 HR cells. These results suggest that Deguelin inhibits growth of both HER-2 overexpressing and those resistant to Herceptin therapy breast cancer cells. Effect of Deguelin appears to be mediated through down regulation of HER-2, PI3K AKT, JAB1, Cyclin D1, and p27 pathway. Our preliminary results warrant further investigation into Deguelin as a potential therapeutic agent for Herceptin resistant breast cancers. (This work was supported by NCI CA140321.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3812. doi:1538-7445.AM2012-3812

Abstract 3659: The AKT inhibitor MK-2206 promotes apoptosis in acute myeloid leukemia cells

Abstract The need for improved therapies for acute myeloid leukemia (AML) is urgent as the disease is still highly fatal. Protein Kinase B (AKT) activation has been shown to predict poor clinical outcome for patients with AML. In 253 newly diagnosed AML cases, we have found that AKT phosphorylation on Thr308 analyzed by reverse-phase protein arrays was significantly higher in patients with unfavorable cytogenetics (p=0.037). The adverse prognostic factor of high levels of pAKT was observed among patients with intermediate-risk cytogenetics (p=0.013), as it was associated with an inferior survival (median survival 44.2 weeks) compared to patients with low or intermediate pAKT (78.7 weeks). Recently, Merck Sharp & Dohme Corp. has developed a compound, MK-2206, that is a highly specific allosteric inhibitor for all three AKT isoforms. In the current study, we have determined that MK-2206 as a single agent used at clinically achievable concentrations suppresses cell growth of a variety of AML derived cell lines including OCI-AML3, U937, and MOLM13. MK-2206 can also induce apoptosis in blast cells derived from AML patients. Consistent with its role as an AKT inhibitor, the drug suppresses phosphorylation of AKT at serine 473 and inhibits phosphorylation of AKT downstream targets including S6 ribosomal protein, 4-EBP1, and PRAS40 in the AML cells. We recently identified that low expression of the PP2A isoform that acts as the AKT phosphatase (i.e. the one containing the B55α subunit) in AML negatively correlates with AKT T308 phosphorylation and activation in a cohort of 511 AML patients (Ruvolo et al Leukemia, 2011). Patients with low B55α expression exhibited shorter complete remission duration. We suppressed B55α expression by shRNA in OCI-AML3 cells to determine if loss of the AKT phosphatase promotes AKT activation and chemoresistance. Cells with reduced B55α exhibited higher levels of AKT phosphorylation and were more resistant to killing by the conventional chemotherapy agent AraC. OCI-AML3 B55α shRNA transductant cells were also more resistant to MK-2206 compared to cells expressing control non-specific shRNA. These results suggest that AKT phosphatase activity in AML may affect response to MK-2206. A recent study in liver cancer demonstrated that MK-2206 synergized with the multi-kinase inhibitor Sorafenib. Since Sorafenib targets the Fms-like tyrosine kinase 3 (FLT3) and mutations that activate the kinase in AML confer poor prognosis, we examined if MK-2206 could synergize with Sorafenib to kill leukemia cells with mutation of FLT3 internal tandem duplication (ITD) mutation. The combination of MK-2206 and Sorafenib was effective at killing MOLM13 cells and AML blast cells with the FLT3-ITD. Taken together, these findings suggest that MK-2206 may be effective to block AKT signaling pathways and could be beneficial as a therapeutic agent perhaps as a single agent or in combination with a drug such as Sorafenib in the therapy of AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3659. doi:1538-7445.AM2012-3659

Abstract 422: Nanog promotes PI3K/Akt and mTOR-mediated invasion in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) affects nearly 40,000 new patients annually in the United States with approximately 50% of these patients having a poor prognosis. Frequent metastasis of the primary tumor to nearby cervical lymph nodes and local critical structures poses a major problem in the treatment of HNSCC. To date, several molecular mediators of invasion and metastasis in HNSCC have been studied; however, with the emergence of the cancer stem cell hypothesis, other genes normally associated with self-renewal and pluripotency in stem cells would also need to be evaluated for their role in tumor invasion and metastasis. In the present study, we used various functional assays to determine the role of Nanog in HNSCC invasion and metastasis as well as identify the underlying pathway through which Nanog acts to regulate invasion and metastasis. We observed that Nanog overexpression increased invasion and cell migration in two cell lines (UMSCC-10B and UMSCC-22B) while Nanog knockdown using siRNA decreased invasion and cell migration in both cell lines and in a putative HNSCC cancer stem cell culture which expressed high levels of Nanog. Treatment with LY294002 or Rapamycin using doses that did not significantly affect cell viability and proliferation dramatically reversed both invasion and migration conferred by Nanog overexpression, indicating the effect is most likely dependent upon the PI3K/Akt/mTOR pathway. By western blot, we found that Nanog-overexpressing cells expressed higher levels of p-Akt and p-mTOR compared to control cells or Nanog-knockdown cells, confirming regulation of Akt by Nanog. In addition, Nanog overexpression resulted in the induction of MMP-2/MMP-9 as well as EMT genes Snail, Vimentin, and Twist. This enhanced expression of EMT was consistent with the observed increased migration as demonstrated by a wound healing assay. Nanog regulation in a mouse model is currently under way to confirm our results in vivo. The results of our study support the possibility of screening for Nanog expression as a prognostic tool. Meanwhile, further efforts aimed at elucidating the signaling pathway between Nanog and Akt may reveal novel treatment targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 422. doi:1538-7445.AM2012-422

PIK3CA/PTEN Mutations and Akt Activation As Markers of Sensitivity to Allosteric mTOR Inhibitors

We sought to determine whether phosphoinositide 3-kinase (PI3K) pathway mutation or activation state and rapamycin-induced feedback loop activation of Akt is associated with rapamycin sensitivity or resistance. Cancer cell lines were tested for rapamycin sensitivity, Akt phosphorylation, and mTOR target inhibition. Mice injected with breast or neuroendocrine cancer cells and patients with neuroendocrine tumor (NET) were treated with rapalogs and Akt phosphorylation was assessed. Thirty-one cell lines were rapamycin sensitive (RS) and 12 were relatively rapamycin resistant (RR; IC(50) > 100 nmol/L). Cells with PIK3CA and/or PTEN mutations were more likely to be RS (P = 0.0123). Akt phosphorylation (S473 and T308) was significantly higher in RS cells (P < 0.0001). Rapamycin led to a significantly greater pathway inhibition and greater increase in p-Akt T308 (P < 0.0001) and p-Akt S473 (P = 0.0009) in RS cells. Rapamycin and everolimus significantly increased Akt phosphorylation but inhibited growth in an in vivo NET model (BON). In patients with NETs treated with everolimus and octreotide, progression-free survival correlated with p-Akt T308 in pretreatment (R = 0.4762, P = 0.0533) and on-treatment tumor biopsies (R = 0.6041, P = 0.0102). Patients who had a documented partial response were more likely to have an increase in p-Akt T308 with treatment compared with nonresponders (P = 0.0146). PIK3CA/PTEN genomic aberrations and high p-Akt levels are associated with rapamycin sensitivity in vitro. Rapamycin-mediated Akt activation is greater in RS cells, with a similar observation in patients with clinical responses on exploratory biomarker analysis; thus feedback loop activation of Akt is not a marker of resistance but rather may function as an indicator of rapamycin activity.

Mutations in FGFR3 and PIK3CA, singly or combined with RAS and AKT1, are associated with AKT but not with MAPK pathway activation in urothelial bladder cancer

Different members of the phosphoinositide 3 kinase--serine threonine protein kinase (PI3K-AKT) pathway are altered in bladder cancer. Fibroblast growth factor receptor 3 (FGFR3) mutations characterize the low-grade tumors, and RAS genes are mutated in approximately 13% of all bladder tumors. Interestingly, a percentage of bladder tumors have alterations in more than 1 PI3K-AKT or rat sarcoma viral oncogene homolog-RAF mitogen activated protein kinase (RAS-MAPK) pathway gene or their upstream regulators, but some combinations are mutually exclusive. We analyzed mutations in FGFR3, phosphoinositide 3 kinase catalytic alpha polypeptide (PIK3CA), v-akt murine thymoma viral oncogene homolog 1 (AKT1), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS), and v-raf murine sarcoma viral oncogene homolog B1 (BRAF) in 88 urothelial cell carcinomas and the immunohistochemical expression of phospho-v-akt murine thymoma viral oncogene homolog (AKT) and mitogen-activated protein kinase 1 and 2 (pERK1/2) in 80 and 77 urothelial cell carcinomas, respectively. Approximately 43% and 20.5% of tumors presented 1 and 2 mutated genes, respectively. FGFR3 mutations were more frequent alone, whereas PIK3CA mutations were associated with another mutated gene (FGFR3 and KRAS). Overall, mutated FGFR3 (FGFR3(mut)) and mutated FGFR3 (FGFR3(mut))-mutated PIK3CA (PIK3CA(mut)) genotypes were associated with low-grade bladder tumors and mutated PIK3CA (PIK3CA(mut))-mutated KRAS (KRAS(mut)) and mutated AKT1 (AKT1(mut)) were only present in high-grade tumors. There are no mutated FGFR3 (FGFR3(mut))-mutated RAS (RAS(mut)) nor mutated PIK3CA (PIK3CA(mut))-mutated AKT1 (AKT1(mut)) combinations. Fifty percent and 56% of tumors showed high levels of pAKT and pERK1/2, respectively. High levels of pAKT were associated with total mutations, FGFR3(mut), and PIK3CA(mut) tumors but not with tumor grade or stage. Wild-type tumors presented significantly higher pERK1/2 expression. Mutations in FGFR3 and FGFR3-PIK3CA but not single PIK3CA mutations characterize low-grade bladder tumors. Single FGFR3 or PIK3CA mutations and the different mutation combinations FGFR3-PIK3CA/AKT1 and PIK3CA-RAS can activate the AKT but not the MAPK pathway. Other genes different from FGFR3 may be related with the pERK activation in bladder tumors.

Expression of pERK and pAKT in pediatric high grade astrocytomas: Correlation with YKL40 and prognostic significance

The Ras signaling pathway, consisting of mitogen-activated protein kinase (MAPK) and PI3K/AKT signaling, is a prominent oncogenic pathways in adult diffuse gliomas, but few studies have evaluated such pathways in pediatric malignant gliomas. We investigated by immunohistochemistry MAPK and AKT signaling in a series of 28 pediatric high-grade gliomas (WHO grade III and IV). We sought a possible association of phospho-ERK (p-ERK) and phospho-AKT (p-AKT) with expression of other proteins involved in the Ras pathway, that is, YKL40, epidermal growth factor receptor (EGFR), EGFR vIII and c-Met. Moreover we correlated the expression of p-ERK and p-AKT with prognosis. No cases showed expression for c-Met and EGFR, and only one case was positive for EGFR vIII. YKL-40 protein was expressed in 43% of cases. We detected expression of p-ERK and p-AKT in 61% and 57%, respectively, of pediatric high grade gliomas. Statistical analysis comparing the two groups in term of high and low p-ERK and p-AKT expression showed a trend toward worse overall survival in patients with high expression of p-AKT. The activation of ERK and AKT suggest a possible role of this protein in inducing activation of the Ras signaling pathway in pediatric high-grade gliomas. Moreover high levels of p-AKT are associated with worse overall survival.

Phospho-ERK and AKT status, but not KRAS mutation status, are associated with outcomes in rectal cancer treated with chemoradiotherapy

BackgroundKRAS mutations may predict poor response to radiotherapy. Downstream events from KRAS, such as activation of BRAF, AKT and ERK, may also confer prognostic information but have not been tested in rectal cancer (RC). Our objective was to explore the relationships of KRAS and BRAF mutation status with p-AKT and p-ERK and outcomes in RC.MethodsPre-radiotherapy RC tumor biopsies were evaluated. KRAS and BRAF mutations were assessed by pyrosequencing; p-AKT and p-ERK expression by immunohistochemistry.ResultsOf 70 patients, mean age was 58; 36% stage II, 56% stage III, and 9% stage IV. Responses to neoadjuvant chemoradiotherapy: 64% limited, 19% major, and 17% pathologic complete response. 64% were KRAS WT, 95% were BRAF WT. High p-ERK levels were associated with improved OS but not for p-AKT. High levels of p-AKT and p-ERK expression were associated with better responses. KRAS WT correlated with lower p-AKT expression but not p-ERK expression. No differences in OS, residual disease, or tumor downstaging were detected by KRAS status.ConclusionsKRAS mutation was not associated with lesser response to chemoradiotherapy or worse OS. High p-ERK expression was associated with better OS and response. Higher p-AKT expression was correlated with better response but not OS.