Supplementary Legends 1-5 and Table 1 Legend from Oxygen Concentration Determines the Biological Effects of NOTCH-1 Signaling in Adenocarcinoma of the Lung

Supplementary Tables 1-2, Figures 1-8 from Opposite Effects of Notch-1 and Notch-2 on Mesothelioma Cell Survival under Hypoxia Are Exerted through the Akt Pathway

Supplementary Figure 2 from Oxygen Concentration Determines the Biological Effects of NOTCH-1 Signaling in Adenocarcinoma of the Lung

Supplementary Figures 1-5, Tables 1-2 from Inhibition of NOTCH Signaling by Gamma Secretase Inhibitor Engages the RB Pathway and Elicits Cell Cycle Exit in T-Cell Acute Lymphoblastic Leukemia Cells

PDF file - 1334KB, S1. IC50 values of TBK1 inhibitors Compound number 5, Compound number 4 and Compound number 3 from the selected PDAC, colorectal cancer and NSCLC cancer cell lines using are shown. Table 1S. List of shRNAs Targeting TBK1 Table 2S. RAS Gene Signature Score Correlation with Sensitivity to the TBK1i (Compound #1) and Meki (PD0325901) in NSCLC Cell Lines Table 3S. List of Cell Lines from 6-Cell Line Panels that were Used in TBK1 Inhibitor Screening in the 2-Dimensional Cell Viability Assay (NSCLC and Colorectal cancer panels) Table 4S. List of Cell Lines from 6-Cell Line Panels that were Used in TBK1 Inhibitor Screening in the 2-Dimensional Cell Viability Assay (Ovarian cancer, GBM and Lymphoma panels).

Supplementary Figure Legends

Supplementary Figures S1, S2. Supplementary Figure 1 shows pharmacodynamic biomarkers for two of the pancreatic cancer subcutaneous patient-derived xenografts. Supplementary Figure 2 shows necropsy, indicating significantly decreased metastases in animals treated with the combination of dinaciclib + MK-2206

Supplementary Figures 1-3, Tables 1-2 from Downregulation of Notch Pathway by a γ-Secretase Inhibitor Attenuates AKT/Mammalian Target of Rapamycin Signaling and Glucose Uptake in an ERBB2 Transgenic Breast Cancer Model

The RAS-MAPK pathway controls many cellular programs, including cell proliferation, differentiation, and apoptosis. In colorectal cancers, recurrent mutations in this pathway often lead to increased cell signaling that may contribute to the development of neoplasms, thereby making this pathway attractive for therapeutic intervention. To this end, we developed a 26-member gene signature of RAS-MAPK pathway activity utilizing the Affymetrix QuantiGene Plex 2.0 reagent system and performed both primary and confirmatory gene expression-based high-throughput screens (GE-HTSs) using KRAS mutant colon cancer cells (SW837) and leveraging a highly annotated chemical library. The screen achieved a hit rate of 1.4% and was able to enrich for hit compounds that target RAS-MAPK pathway members such as MEK and EGFR. Sensitivity and selectivity performance measurements were 0.84 and 1.00, respectively, indicating high true-positive and true-negative rates. Active compounds from the primary screen were confirmed in a dose-response GE-HTS assay, a GE-HTS assay using 14 additional cancer cell lines, and an in vitro colony formation assay. Altogether, our data suggest that this GE-HTS assay will be useful for larger unbiased chemical screens to identify novel compounds and mechanisms that may modulate the RAS-MAPK pathway.

γ‐Secretase mediates amyloid production in Alzheimer's disease (AD) and oncogenic activity of Notch. γ‐Secretase inhibitors (GSIs) are thus of interest for AD and oncology. A peripheral biomarker of Notch activity would aid determination of the therapeutic window and dosing regimen for GSIs, given toxicities associated with chronic Notch inhibition. This study examined the effects of GSI MK‐0752 on blood and hair follicle transcriptomes in healthy volunteers. The effects of a structurally diverse GSI on rhesus blood and hair follicles were also compared. Significant dose‐related effects of MK‐0752 on transcription were observed in hair follicles, but not blood. The GSI biomarker identified in follicles exhibited 100% accuracy in a clinical test cohort, and was regulated in rhesus by a structurally diverse GSI. This study identified a translatable, accessible pharmacodynamic biomarker of GSI target engagement and provides proof of concept of hair follicle RNA as a translatable biomarker source.

Abstract KRAS is activated by mutation in the vast majority of cases of pancreatic cancer; unfortunately, therapeutic attempts to inhibit KRAS directly have been unsuccessful. Our previous studies showed that inhibition of cyclin-dependent kinase 5 (CDK5) reduces pancreatic cancer growth and progression, through blockage of the centrally important RAL effector pathway, downstream of KRAS. In the current study, the therapeutic effects of combining the CDK inhibitor dinaciclib (SCH727965; MK-7965) with the pan-AKT inhibitor MK-2206 were evaluated using orthotopic and subcutaneous patient-derived human pancreatic cancer xenograft models. The combination of dinaciclib (20 mg/kg, i.p., three times a week) and MK-2206 (60 mg/kg, orally, three times a week) dramatically blocked tumor growth and metastasis in all eight pancreatic cancer models examined. Remarkably, several complete responses were induced by the combination treatment of dinaciclib and MK-2206. The striking results obtained in these models demonstrate that the combination of dinaciclib with the pan-AKT inhibitor MK-2206 is promising for therapeutic evaluation in pancreatic cancer, and strongly suggest that blocking RAL in combination with other effector pathways downstream from KRAS may provide increased efficacy in pancreatic cancer. Based on these data, an NCI–CTEP-approved multicenter phase I clinical trial for pancreatic cancer of the combination of dinaciclib and MK-2206 (NCT01783171) has now been opened. Mol Cancer Ther; 14(7); 1532–9. ©2015 AACR.

Glucocorticoid signaling regulates target genes by multiple mechanisms, including the repression of transcriptional activities of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) though direct protein-protein interactions and subsequent O-GlcNAcylation of RNA polymerase II (pol II). Recent studies have shown that overexpression of O-linked β-N-acetylglucosamine transferase (OGT), which adds an O-linked β-N-acetylglucosamine (O-GlcNAc) group to the C-terminal domain of RNA pol II, increases the transrepression effects of glucocorticoids (GC). As O-GlcNAcase (OGA) is an enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, we hypothesized that the potentiation of GC effects following OGT overexpression could be similarly observed via the direct inhibition of OGA, inhibiting O-GlcNAc removal from pol II. Here we show that despite pharmacological evidence of target engagement by a selective small molecule inhibitor of OGA, there is no evidence for a sensitizing effect on glucocorticoid-mediated effects on TNF-α promoter activity, or gene expression generally, in human cells. Furthermore, inhibition of OGA did not potentiate glucocorticoid-induced apoptosis in several cancer cell lines. Thus, despite evidence for O-GlcNAc modification of RNA pol II in GR-mediated transrepression, our data indicate that pharmacological inhibition of OGA does not potentiate or enhance glucocorticoid-mediated transrepression.

TBK1 (TANK-binding kinase 1) is a noncanonical IkB protein kinase that phosphorylates and activates downstream targets such as IRF3 and c-Rel and, mediates NF-kB activation in cancer. Previous reports demonstrated synthetic lethality of TBK1 with mutant KRAS in non–small cell lung cancer (NSCLC); thus, TBK1 could be a novel target for treatment ofKRAS-mutantNSCLC.Here, the effect of TBK1 onproliferation in a panel of cancer cells by both genetic and pharmacologic approaches was evaluated. In KRAS-mutant cancer cells, reduction of TBK1 activity by knockdown or treatment with TBK1 inhibitors did not correlate with reduced proliferation in a two-dimensional viability assay. Verification of target engagement via reduced phosphorylation of S386 of IRF3 (pIRF3) was difficult to assess inNSCLCcells due to lowprotein expression.However, several cell lines were identified with high pIRF3 levels after screening a large panel of cell lines, many of which also harbor KRASmutations. Specifically, a large subset of KRAS-mutant pancreatic cancer cell lines was uncovered with high constitutive pIRF3 levels, which correlated with high levels of phosphorylated S172 of TBK1 (pTBK1). Finally, TBK1 inhibitors dose-dependently inhibited pIRF3 in these cell lines, but this did not correlate with inhibition of cell growth. Taken together, these data demonstrate that the regulation of pathways important for cell proliferation in some NSCLC, pancreatic, and colorectal cell lines is not solely dependent on TBK1 activity. Implications:TBK1has therapeutic potential under certain contexts and phosphorylation of its downstream target IRF3 is a biomarker of TBK1 activity. Visual Overview: http://mcr.aacrjournals.org/content/12/7/1055/F1.large.jpg. Mol Cancer Res; 12(7); 1055–66. 2014 AACR.

Dinaciclib is a potent CDK1, 2, 5 and 9 inhibitor being developed for the treatment of cancer. Additional understanding of antitumor mechanisms and identification of predictive biomarkers are important for its clinical development. Here we demonstrate that while dinaciclib can effectively block cell cycle progression, in vitro and in vivo studies, coupled with mouse and human pharmacokinetics, support a model whereby induction of apoptosis is a main mechanism of dinaciclib's antitumor effect and relevant to the clinical duration of exposure. This was further underscored by kinetics of dinaciclib-induced downregulation of the antiapoptotic BCL2 family member MCL1 and correlation of sensitivity with the MCL1-to-BCLxL mRNA ratio or MCL1 amplification in solid tumor models in vitro and in vivo. This MCL1-dependent apoptotic mechanism was additionally supported by synergy with the BCL2, BCL-xL and BCL-w inhibitor navitoclax (ABT-263). These results provide the rationale for investigating MCL1 and BCL-xL as predictive biomarkers for dinaciclib antitumor response and testing combinations with BCL2 family member inhibitors.

Abstract TBK1 (TANK-binding kinase 1) is a noncanonical IκB protein kinase that phosphorylates and activates downstream targets such as IRF3 and c-Rel and, mediates NF-κB activation in cancer. Previous reports demonstrated synthetic lethality of TBK1 with mutant KRAS in non–small cell lung cancer (NSCLC); thus, TBK1 could be a novel target for treatment of KRAS-mutant NSCLC. Here, the effect of TBK1 on proliferation in a panel of cancer cells by both genetic and pharmacologic approaches was evaluated. In KRAS-mutant cancer cells, reduction of TBK1 activity by knockdown or treatment with TBK1 inhibitors did not correlate with reduced proliferation in a two-dimensional viability assay. Verification of target engagement via reduced phosphorylation of S386 of IRF3 (pIRF3S386) was difficult to assess in NSCLC cells due to low protein expression. However, several cell lines were identified with high pIRF3S386 levels after screening a large panel of cell lines, many of which also harbor KRAS mutations. Specifically, a large subset of KRAS-mutant pancreatic cancer cell lines was uncovered with high constitutive pIRF3S386 levels, which correlated with high levels of phosphorylated S172 of TBK1 (pTBK1S172). Finally, TBK1 inhibitors dose-dependently inhibited pIRF3S386 in these cell lines, but this did not correlate with inhibition of cell growth. Taken together, these data demonstrate that the regulation of pathways important for cell proliferation in some NSCLC, pancreatic, and colorectal cell lines is not solely dependent on TBK1 activity. Implications: TBK1 has therapeutic potential under certain contexts and phosphorylation of its downstream target IRF3 is a biomarker of TBK1 activity. Visual Overview: http://mcr.aacrjournals.org/content/12/7/1055/F1.large.jpg. Mol Cancer Res; 12(7); 1055–66. ©2014 AACR.

Homologous recombination (HR) is a cellular mechanism for accurate repair of double-strand DNA breaks, often deregulated in cancer. Development of novel cancer therapeutics targeting HR pathways would benefit from a quantitative and rapid means of measuring HR. Here, we describe a zinc finger nuclease (ZFN) assay that can quantify HR. Knockdown of BRCA1 or inactivation of BRCA2 decreased HR activity in cells, whereas gene restoration induced activity 8-fold. HR activity was also reflective of BRCA1/2 status in cells with known endogenous mutations.

Abstract TBK1 is a non-canonical IκB protein kinase that phosphorylates and activates downstream targets such as IRF3 and cRel and, reportedly mediates NFkB activation in cancer. We used both genetic and pharmacological approaches to evaluate TBK1 function in cancer cells. In oncogenic KRAS mutant lung cancer cell lines, the TBK1 knockdown did not correlate with reduction in a 2D viability assay. Using TBK1 small molecule inhibitors, we found that the inhibition of cell growth in 2D viability assays did not correlate with Kras dependency or RAS genetic signature. Since the pIRF3(S386) levels in lung lines were too low to assess TBK1 target engagement, we identified cell lines with high pIRF3(S386) levels. We discovered a subset of pancreatic and colorectal cell lines that have high constitutive pIRF3(S386) levels and this correlated with high levels of pTBK1(S172). TBK1 inhibitors were able to dose-dependently inhibit pIRF3S386 in pancreatic & colon lines. These observations indicate that pIRF3(S386) could be used as a biomarker for direct target engagement of TBK1 in cancer cell lines with activated TBK1 pathway. pTBK1(S172), a phosphorylation site within the TBK1 kinase domain activation loop, does not decrease upon inhibition with TBK1 inhibitors but, instead increases. This suggests stimulation of a feedback loop resulting in the activation of an upstream kinase(s) responsible for activating TBK1 in cancer cell lines. Although several pancreatic and colon cell lines were identified to have low μM sensitivity to TBK1 inhibitors, TBK1 shRNA knockdown did not impact proliferation in these cell lines even though pIRF3(S386) levels were reduced. Further studies using knockdown of the related IKKe homolog in cell lines with activated pathway are also being pursued to determine if IKKe knockdown alone or in combination with TBK1 knockdown would result in more substantial growth reduction. It is likely that inhibition of additional nodes in the NF-kB pathway and other pathways are needed to achieve robust anti-tumor efficacy. Citation Format: Asli Muvaffak, Qi Pan, Richard Middleton, Erick Morris, Rafael Fernandez, Jonwong Lim, Brian Dolinski, Thi T. Nguyen, Peter Strack, Haiyan Yan, Rossana Chung, Stephen Wu, Weiqun Zhang, Chris Hulton, Heather Hirsch, Kumiko Nagashimo, Yan Wang. Evaluating TBK1 as a cancer therapeutic target in cancer cell lines with activated IRF3. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4372. doi:10.1158/1538-7445.AM2013-4372

Abstract Dinaciclib is a novel CDK1, 2, 5, and 9 inhibitor currently in clinical development for CLL. Preclinical studies indicate that dinaciclib may have activity in a wide variety of indications including hematologic malignancies and solid tumors. Clinically, dinaciclib is administered by 2 hour intravenous (iv) infusion and achieves plasma concentrations above 100nM for ∼6 hours; a concentration and duration of treatment which in vitro provides complete target engagement and induces apoptosis. Pre-clinically, dinaciclib is administered by intraperitoneal (ip) bolus injections and achieves plasma concentrations above 100nM for less than 2 hours. To determine whether prolonged plasma concentrations would enhance dinaciclib activity, we administered dinaciclib to COLO-320DM tumor bearing mice either as a 40 mg/kg single dose, or as two doses of 20 mg/kg separated by 2 hours. This 20-20 split dosing resulted in prolonged target engagement, phospho-RNA-Polymerase-2 reduction, decreased MCL-1, and increased apoptosis relative to the 40mg/kg single dose. Comparing the in vivo anti-tumor activity of dinaciclib dosed at 40 mg/kg ip every 4 days (q4d) vs. 20 mg/kg bid separated by 2 hours q4d demonstrated that the 20-20 split dose increased the anti-tumor activity of dinaciclib (25% Tumor Growth Inhibition (TGI) versus 66% TGI). Dinaciclib single vs. split dosing was further evaluated in 8 lung cancer xenograft models, 5 of which were insensitive to dinaciclib single dose (TGI >50%) and 3 of which were sensitive. 20-20 split dosing resulted in increased anti-tumor activity in all models tested. Split dosing resulted in <50% TGI in 4 of 5 insensitive models. Split dosing also increased anti-tumor activities in all 3 sensitive models tested resulting in stasis in 2 models, and sustained complete regressions in one lung cancer xenograft model. Body weight measurements revealed that split dosing increased body weight loss indicating that prolonged pathway inhibition can affect both anti-tumor activity and tolerability. Taken together, these data suggest that dinaciclib dosing paradigms that prolong pathway inhibition enhanced anti-tumor activity in pre-clinical xenograft models. Citation Format: Alwin Schuller, Robert Booher, Louise Cadzow, Minilik Angagaw, Lauren Harmonay, Xianlu Qu, Nathan Miselis, Vincenzo Pucci, Mark Ayers, Thorsten Graef, Ellie Im, Rebecca Blanchard, Brian Long, Leigh Zawel, peter strack. Optimized dosing strategies resulting in prolonged pathway inhibition enhance dinaciclib anti-tumor activity in preclinical xenograft models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 699. doi:10.1158/1538-7445.AM2013-699

Abstract Pancreatic cancer is among the deadliest of all solid malignances. The 5-year survival rate of pancreatic cancer remains below 5%, mainly due to a lack of early detection and the highly metastatic properties that make surgery impossible in most cases. Therefore, it is urgent to find effective systemic therapies to treat this highly metastatic cancer. KRAS is activated in the vast majority of cases of pancreatic cancer; unfortunately, therapeutic attempts to inhibit KRAS directly have been unsuccessful. Our previous studies showed that inhibition of cyclin-dependent kinase 5 (CDK5), using genetic manipulation or the CDK inhibitor Dinaciclib, reduces pancreatic cancer growth and progression, through blockage of the Ral effector pathway, downstream of KRAS. Since Ral has been shown to be a centrally important effector of KRAS signaling in pancreatic cancer, we hypothesized that simultaneous blockage of two major downstream KRAS effectors may further inhibit the growth and metastasis of pancreatic cancer and provide an effective therapeutic strategy for this disease. In the current study, the therapeutic effects of combining the CDK inhibitor Dinaciclib with inhibitors of either the PI3K/AKT effector pathway (pan-AKT inhibitor MK2206) or the RAF/MEK/ERK effector pathway (ERK inhibitor SCH772984) were evaluated using two orthotopic patient-derived human pancreatic cancer xenograft models (Panc253 and Panc265). These models closely resemble the physiological and pathological conditions of pancreatic cancer in humans. A 2-3 mm3 tumor explant was implanted into the pancreas of nude mice and ultrasound imaging was used to measure the tumor size (3D) before randomization and treatment, which began when tumors grew to 50-100 mm3. The combination of Dinaciclib (20 mg/kg, i.p., t.i.w.) and MK2206 (60 mg/kg, p.o., t.i.w.) dramatically blocked tumor growth in the orthotopic Panc265 (90.0%, p<0.001) and Panc253 (93.0%, p<0.001) models versus control. It also significantly reduced the number of metastatic lesions in both Panc265 (88.2%, p<0.001) and Panc253 (99.0%, p<0.001) tumor models. Remarkably, complete responses were induced by the combination treatment of Dinaciclib and MK2206 in one mouse in the Panc265 tumor model, and in two mice in the Panc253 tumor model. Similarly, the combination of Dinaciclib (20 mg/kg, i.p., t.i.w.) and SCH772984 (25 mg/kg, i.p., b.i.d.) dramatically inhibited the growth of primary orthotopic Panc265 (82.5%, p < 0.001) and Panc253 (95.7%, p <0.001) versus control, and also the number of metastatic lesions of both Panc265 (94.9%, p <0.001) and Panc253 (92.4%, p <0.02). The striking results obtained in these models appear to validate our hypothesis that blocking multiple effector pathways downstream from KRAS, including the Ral effector pathway, may provide increased efficacy in pancreatic cancer. Thus, the combinations of Dinaciclib with the pan-AKT inhibitor MK2206 or the ERK inhibitor SCH772984 are novel, highly promising potential therapies against pancreatic cancer. Based on these data, an NCI-CTEP approved Phase I clinical trial for pancreatic cancer of the combination of Dinaciclib and MK2206 has now opened here at Johns Hopkins. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B263. Citation Format: Chaoxin Hu, Tikva Dadon, Venugopal Chenna, Shin Yabuuchi, Rajat Bannerji, Robert Booher, Peter Strack, Ahmed Samatar, Nilofer Azad, Barry D. Nelkin, Anirban Maitra. Combined inhibition of cyclin-dependent kinases (Dinaciclib) and AKT (MK-2206) or ERK (SCH772984) dramatically blocks pancreatic tumor growth and metastases in patient-derived orthotopic xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B263.