K-RAS Mutant Pancreatic Tumors Show Higher Sensitivity to MEK than to PI3K Inhibition <em>In Vivo</em>

<div><p>Activating K-RAS mutations occur at a frequency of 90% in pancreatic cancer, and to date no therapies exist targeting this oncogene. K-RAS signals via downstream effector pathways such as the MAPK and the PI3K signaling pathways, and much effort has been focused on developing drugs targeting components of these pathways. To better understand the requirements for K-RAS and its downstream signaling pathways MAPK and PI3K in pancreatic tumor maintenance, we established an inducible K-RAS knock down system that allowed us to ablate K-RAS in established tumors. Knock down of K-RAS resulted in impaired tumor growth in all pancreatic xenograft models tested, demonstrating that K-RAS expression is indeed required for tumor maintenance of K-RAS mutant pancreatic tumors. We further examined signaling downstream of K-RAS, and detected a robust reduction of pERK levels upon K-RAS knock down. In contrast, no effect on pAKT levels could be observed due to almost undetectable basal expression levels. To investigate the requirement of the MAPK and the PI3K pathways on tumor maintenance, three selected pancreatic xenograft models were tested for their response to MEK or PI3K inhibition. Tumors of all three models regressed upon MEK inhibition, but showed less pronounced response to PI3K inhibition. The effect of MEK inhibition on pancreatic xenografts could be enhanced further by combined application of a PI3K inhibitor. These data provide further rationale for testing combinations of MEK and PI3K inhibitors in clinical trials comprising a patient population with pancreatic cancer harboring mutations in K-RAS.</p> </div

Discriminating Survival Outcomes in Patients with Glioblastoma Using a Simulation-Based, Patient-Specific Response Metric

<div><p>Accurate clinical assessment of a patient's response to treatment for glioblastoma multiforme (GBM), the most malignant type of primary brain tumor, is undermined by the wide patient-to-patient variability in GBM dynamics and responsiveness to therapy. Using computational models that account for the unique geometry and kinetics of individual patients' tumors, we developed a method for assessing treatment response that discriminates progression-free and overall survival following therapy for GBM. Applying these models as untreated virtual controls, we generate a patient-specific “Days Gained” response metric that estimates the number of days a therapy delayed imageable tumor progression. We assessed treatment response in terms of Days Gained scores for 33 patients at the time of their first MRI scan following first-line radiation therapy. Based on Kaplan-Meier analyses, patients with Days Gained scores of 100 or more had improved progression-free survival, and patients with scores of 117 or more had improved overall survival. Our results demonstrate that the Days Gained response metric calculated at the routinely acquired first post-radiation treatment time point provides prognostic information regarding progression and survival outcomes. Applied prospectively, our model-based approach has the potential to improve GBM treatment by accounting for patient-to-patient heterogeneity in GBM dynamics and responses to therapy.</p> </div

GDC0941 and AZD6244 <i>in vivo</i> treatment inhibits pAKT and pERK respectively.

<p>Indicated tumor-bearing mice were treated with a single dose of either GDC0941 100 mg/kg p.o., or AZD6244 50 mg/kg p.o., or with vehicle control. Animals were sacrificed 1 h after treatment, plasma samples were collected, analyzed and quantified by mass spectrometry for GDC0941 (A) or AZD6244 (B). Tumors were excised and analyzed by Western Blot for total AKT, pAKT (Ser473), total ERK or pERK (Thr202/Tyr204) for the model Rat1-myr-p110α (C) or the model Panc 10.05 (D).</p

K-RAS knock down impairs tumor growth of pancreatic models <i>in vivo</i>.

<p>(A) For each xenograft model indicated, tumors were grown subcutaneously in female nude mice and groups of at least 4 mice each were formed once tumors had reached a size of 200–300 mm³. The first group was given normal drinking water, whereas the second was given drinking water containing 2 mg/ml doxycycline and 10% sucrose. Mice were sacrificed after one week of treatment (after 18 days in case of the K-RAS wild type model), and tumors were analyzed by qPCR for K-RAS. K-RAS levels were normalized to ribosomal protein s18. Obtained p-values were as follows: Capan-1 shNT: p = 0.35, Capan-1 sh236: p = 0.011, Panc 10.05 sh236: p = 0.009, AsPC-1 sh236: p = 0.002, L3.3 sh236: p = 0.004, NCI-H1437 sh236: p = 0.007. (B/C) As in (A), except that mice were randomized to groups of at least 6 mice each, with the exception of the Panc 10.05 model, where the group size was n = 4. Treatment was started once tumors had reached a size of 100 mm³, tumor size was followed over time, and mice were sacrificed once tumors of the control group reached a size of 1000 mm³ at most. Statistically significant differences of tumor volumes between groups (*) as well as the area under the curve (AUC/mm³ x treatment days) are indicated. Obtained p-values for AUC at the end of the study were as follows: Capan-1 shNT: p = 0.57, Capan-1 sh236: p = 0.04, Panc 10.05 sh236: p = 0.01, AsPC-1 sh236: p = 0.01, L3.3 sh236: p = 0.0003, NCI-H1437: p = 0.22. The PANC-1 cells could not be grown <i>in vivo</i>, and for this reason this model was only examined <i>in vitro</i>.</p

K-RAS mutant pancreatic models show stronger response to MEK than to PI3K inhibition <i>in vivo</i>.

<p>(A/B). Indicated tumor-bearing mice were treated either with GDC0941 100 mg/kg p.o. once a day, or with AZD6244 50 mg/kg p.o. twice a day, or with vehicle control, with at least 5 mice per group. Tumor volumes were measured twice a week for the indicated period of time, and antitumor activity was plotted and quantified.</p

K-RAS knock down results in decreased pERK levels <i>in vivo</i>.

<p>For each xenograft model indicated, tumors were grown subcutaneously in female nude mice and groups of at least 4 mice each were formed once tumors had reached a size of 200–300 mm³. The first group was given normal drinking water (-dox), whereas the second was given drinking water containing 2 mg/ml doxycycline and 10% sucrose (+dox). After one week of treatment, mice were sacrificed and the tumors were removed and processed for immunohistochemistry for either pERK (Thr202/Tyr204) (A), or pAKT (Ser473) (B). The T47D model was used as an AKT dependent control model with physiological pAKT levels.</p

K-RAS knock down impairs proliferation in pancreatic lines <i>in vitro</i>.

<p>(A) Indicated cell lines (NT: non-targeting shRNA; 236 and 562: shRNAs targeting K-RAS) were either treated for 7 days with 200 ng/ml doxycycline (dox) or left untreated (no dox), followed by preparation of cell lysates. Corresponding cell extracts were then analyzed for K-RAS, total AKT, pAKT (Ser473), total ERK or pERK (Thr202/Tyr204) levels by Western Blot. (B) As in (A), except that cells were fixed on day 1 and day 7, followed by determination of relative cell number. Each cell line was tested in at least two independent experiments, and untreated samples were set to 100% of growth. Statistically significant differences (p<0.05) are indicated (*). Obtained p-values were as follows: Capan-1 shNT: p = 1, Capan-1 sh236: p = 0.002, Capan-1 sh562: p = 0.029; Panc 10.05 shNT: p = 0.33, Panc 10.05 sh236: p<0.001, Panc 10.05 sh562: p = 0.029; AsPc1 shNT: p = 0.33, AsPc1 sh236: p = 0.002, AsPc1 sh562: p = 0.029; L3.3 shNT: p = 0.187, L3.3 sh236: p<0.001, L3.3 sh562: p = 0.333; PANC-1 shNT: p = 1, PANC-1 sh236: p<0.001, PANC-1 sh562: p = 0.002.</p

K-RAS mutant pancreatic lines are independent of AKT <i>in vitro</i>.

<p>(A). Indicated cell lines were treated for 72 h with the AKT inhibitor MK2206, and effects on proliferation were determined by calculation of respective GI₅₀ values. (B/C). As in (A), except that indicated cell lines were treated for 62 h with either the PI3K inhibitor GDC0941 (B) or with the MEK inhibitor AZD6244 (C), and effects on proliferation were determined by calculation of respective GI₅₀ values. MCF7 cells were used as control for cells sensitive to GDC0941 and insensitive to AZD6244 and A375 cells were used as control for cells sensitive to AZD6244 and insensitive to GDC0941.</p

Combining MEK and PI3K inhibition <i>in vivo</i> is superior to single agent treatment.

<p>(A). Indicated tumor-bearing mice were treated either with GDC0941 100 mg/kg p.o. once a day, or with AZD6244 5 mg/kg p.o. once a day, or with the combination of both, or with vehicle control, with 8 mice per group. Tumor volumes were measured twice a week, for the indicated period of time, and antitumor activity was plotted and quantified. (B). Indicated tumor-bearing mice were treated with a single dose of either GDC0941 100 mg/kg p.o. or of AZD6244 5 mg/kg p.o., with the combination of both or with vehicle control. Animals were sacrificed 3 h after treatment, tumors were excised and analyzed by Western Blot for total AKT, pAKT (Ser473), total ERK or pERK (Thr202/Tyr204).</p

Kaplan-Meier analyses on progression-free and overall survival.

<p><i>a</i>: Analysis on progression-free survival data revealed a significant difference between the patients with Days Gained scores greater than or equal to 100 and those with lower scores. <i>b</i>: Overall survival analysis also revealed a significant difference between the patients with Days Gained scores greater than or equal to 117 and those with lower scores.</p