Olga K. Mirzoeva

Basal Subtype and MAPK/ERK Kinase (MEK)-Phosphoinositide 3-Kinase Feedback Signaling Determine Susceptibility of Breast Cancer Cells to MEK Inhibition

Specific inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) have been developed that efficiently inhibit the oncogenic RAF-MEK-ERK pathway. We used a systems-based approach to identify breast cancer subtypes particularly susceptible to MEK inhibitors and to understand molecular mechanisms conferring resistance to such compounds. Basal-type breast cancer cells were found to be particularly susceptible to growth inhibition by small-molecule MEK inhibitors. Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in response to MEK inhibition through a negative MEK-epidermal growth factor receptor-PI3K feedback loop was found to limit efficacy. Interruption of this feedback mechanism by targeting MEK and PI3K produced synergistic effects, including induction of apoptosis and, in some cell lines, cell cycle arrest and protection from apoptosis induced by proapoptotic agents. These findings enhance our understanding of the interconnectivity of oncogenic signal transduction circuits and have implications for the design of future clinical trials of MEK inhibitors in breast cancer by guiding patient selection and suggesting rational combination therapies.

Akt activation suppresses Chk2-mediated, methylating agent-induced G2 arrest and protects from temozolomide-induced mitotic catastrophe and cellular senescence.

Pharmacologic inhibition of the DNA signal transducers Chk1 and p38 blocks G2 arrest and sensitizes glioblastoma cells to chemotherapeutic methylating agent-induced cytotoxicity. Because Akt pathway activation has been suggested to also block G2 arrest induced by DNA-damaging agents and because glioma cells frequently have high levels of Akt activation, we examined the contribution of the Akt pathway to methylating agent-induced G2 arrest and toxicity. U87MG human glioma cells containing an inducible Akt expression construct were incubated with inducing agent or vehicle, after which the cells were exposed to temozolomide and assayed for activation of the components of the G2 arrest pathway and survival. Temozolomide-treated control cells activated the DNA damage signal transducers Chk1, Chk2, and p38, leading to Cdc25C and Cdc2 inactivation, prolonged G2 arrest, and loss of clonagenicity by a combination of senescence and mitotic catastrophe. Temozolomide-treated cells induced to overexpress Akt, however, exhibited significantly less drug-induced Cdc25C/Cdc2 inactivation and less G2 arrest. Akt-mediated suppression of G2 arrest was associated not with alterations in Chk1 or p38 activation but rather with suppression of Chk2 activation and reduced recruitment of Chk2 to sites of damage in chromatin. Unlike bypass of the G2 checkpoint induced by pharmacologic inhibitors of Chk1 or p38, however, Akt-induced bypass of G2 arrest suppressed, rather than enhanced, temozolomide-induced senescence and mitotic catastrophe. These results show that whereas Akt activation suppresses temozolomide-induced Chk2 activation and G2 arrest, the overriding effect is protection from temozolomide-induced cytotoxicity. The Akt pathway therefore represents a new target for the sensitization of gliomas to chemotherapeutic methylating agents such as temozolomide.

Autophagy suppression promotes apoptotic cell death in response to inhibition of the PI3K—mTOR pathway in pancreatic adenocarcinoma

Targeting of pathways downstream of RAS represents a promising therapeutic strategy for pancreatic cancer, the fourth leading cause of cancer-related death in the USA, since activation of the Raf-MEK-ERK and PI3K-AKT pathways is found frequently in this disease and is associated with poor prognosis. Taking advantage of a panel of human PDAC cell lines and specific inhibitors of PI3K and/or mTOR, we systematically address the question whether dual-targeted inhibition of the PI3K and mTOR pathways offers advantages over single-targeted inhibition of PI3K in PDAC. We observe greater overall susceptibility of cell lines to dual inhibition compared to targeting PI3K alone. However, we find that dual inhibition of PI3K and mTOR induces autophagy to a greater extent than inhibition of each target alone. In agreement with this, we show that combined administration of PI3K/mTOR and autophagy inhibitors results in increased anti-tumor activity in vitro and in vivo in models of pancreatic adenocarcinoma. XL765, a PI3K/mTOR inhibitor used in our in vivo studies, is currently undergoing clinical evaluation in a variety of cancer types, while the autophagy inhibitor chloroquine is a widely used anti-malaria compound. Thus, our studies provide rationale for clinical development of combinations of these compounds for the treatment of pancreatic adenocarcinoma.

The Mre11/Rad50/Nbs1 complex interacts with the mismatch repair system and contributes to temozolomide-induced G2 arrest and cytotoxicity

The chemotherapeutic agent temozolomide produces O6-methylguanine (O6MG) in DNA, which triggers futile DNA mismatch repair, DNA double-strand breaks (DSB), G2 arrest, and ultimately cell death. Because the protein complex consisting of Mre11/Rad50/Nbs1 (MRN complex) plays a key role in DNA damage detection and signaling, we asked if this complex also played a role in the cellular response to temozolomide. Temozolomide exposure triggered the assembly of MRN complex into chromatin-associated nuclear foci. MRN foci formed significantly earlier than γ-H2AX and 53BP1 foci that assembled in response to temozolomide-induced DNA DSBs. MRN foci formation was suppressed in cells that incurred lower levels of temozolomide-induced O6MG lesions and/or had decreased mismatch repair capabilities, suggesting that the MRN foci formed not in response to temozolomide-induced DSB but rather in response to mismatch repair processing of mispaired temozolomide-induced O6MG lesions. Consistent with this idea, the MRN foci colocalized with those of proliferating cell nuclear antigen (a component of the mismatch repair complex), and the MRN complex component Nbs1 coimmunoprecipitated with the mismatch repair protein Mlh1 specifically in response to temozolomide treatment. Furthermore, small inhibitory RNA–mediated suppression of Mre11 levels decreased temozolomide-induced G2 arrest and cytotoxicity in a manner comparable to that achieved by suppression of mismatch repair. These data show that temozolomide-induced O6MG lesions, acted upon by the mismatch repair system, drive formation of the MRN complex foci and the interaction of this complex with the mismatch repair machinery. The MRN complex in turn contributes to the control of temozolomide-induced G2 arrest and cytotoxicity, and as such is an additional determining factor in glioma sensitivity to DNA methylating chemotherapeutic drugs such as temozolomide. [Mol Cancer Ther 2006;5(11):2757–66]

Subtype-Specific MEK-PI3 Kinase Feedback as a Therapeutic Target in Pancreatic Adenocarcinoma

Mutations in the KRAS oncogene are dominant features in pancreatic ductal adenocarcinoma (PDA). Because KRAS itself is considered “undruggable,” targeting pathways downstream of KRAS are being explored as a rational therapeutic strategy. We investigated the consequences of MAP–ERK kinase (MEK) inhibition in a large PDA cell line panel. Inhibition of MEK activated phosphoinositide 3-kinase in an EGF receptor (EGFR)-dependent fashion and combinations of MEK and EGFR inhibitors synergistically induced apoptosis. This combinatorial effect was observed in the epithelial but not mesenchymal subtype of PDA. RNA expression analysis revealed predictors of susceptibility to the combination, including E-cadherin, HER3, and the miR200-family of microRNAs, whereas expression of the transcription factor ZEB1 was associated with resistance to the drug combination. Knockdown of HER3 in epithelial-type and ZEB1 in mesenchymal-type PDA cell lines resulted in sensitization to the combination of MEK and EGFR inhibitors. Thus, our findings suggest a new, subtype-specific, and personalized therapeutic strategy for pancreatic cancer. Mol Cancer Ther; 12(10); 2213–25. ©2013 AACR.

Bayesian network inference modeling identifies TRIB1 as a novel regulator of cell-cycle progression and survival in cancer cells

Molecular networks governing responses to targeted therapies in cancer cells are complex dynamic systems that demonstrate nonintuitive behaviors. We applied a novel computational strategy to infer probabilistic causal relationships between network components based on gene expression. We constructed a model comprised of an ensemble of networks using multidimensional data from cell line models of cell-cycle arrest caused by inhibition of MEK1/2. Through simulation of a reverse-engineered Bayesian network model, we generated predictions of G1-S transition. The model identified known components of the cell-cycle machinery, such as CCND1, CCNE2, and CDC25A, as well as revealed novel regulators of G1-S transition, IER2, TRIB1, TRIM27. Experimental validation of model predictions confirmed 10 of 12 predicted genes to have a role in G1-S progression. Further analysis showed that TRIB1 regulated the cyclin D1 promoter via NFκB and AP-1 sites and sensitized cells to TRAIL-induced apoptosis. In clinical specimens of breast cancer, TRIB1 levels correlated with expression of NFκB and its target genes (IL8, CSF2), and TRIB1 copy number and expression were predictive of clinical outcome. Together, our results establish a critical role of TRIB1 in cell cycle and survival that is mediated via the modulation of NFκB signaling. Cancer Res; 77(7); 1575-85. ©2017 AACR.

Abstract 5244: Next-generation sequence analysis of cell-free DNA in patients with chemotherapy-refractory advanced pancreatic adenocarcinoma (PDAC) treated with selumetinib (AZD6244) and erlotinib

Background: Monitoring molecular events in response to treatment with targeted agents is particularly challenging in pancreatic cancer due to its poor accessibility for biopsies and high stroma content. Recent advances in digital sequencing of rare DNA species allows for detection of circulating tumor-derived free DNA (ctDNA) in plasma of patients with cancer. Here, we explored the potential of gaining information on the genomic composition of pancreatic cancers in patients treated with a combination of the MEK inhibitor selumetinib (AZD6244) and the EGFR kinase inhibitor, erlotinib. Methods: 46 patients were enrolled into this study at two study centers. Treatment consisted of erlotinib 100 mg + AZD6244 100 mg daily in 3-week cycles, with tumor evaluation by CT scan every 2 cycles. Primary objective was overall survival (OS). Correlative endpoints included assessment of expression of EMT-related proteins, and next-generation sequencing analysis of ctDNA. Plasma “on treatment” samples were uniformly obtained at the 6 week time point. Results: Our preclinical studies had demonstrated that inhibition of MEK leads to enhanced signaling through EGFR with hyperactivation of a parallel RAS effector pathway (PI3K), supporting a therapeutic strategy of combined target inhibition in PDAC to overcome this negative feedback loop. Disease control rate was 58% (0 PR; 19 with stable disease (SD) > 6 weeks, 12 with SD > 12 weeks, including 12 (26%) minor responses). 13/34 patients (38%) demonstrated CA19-9 decline > 50%. Assessment of E-cadherin expression in pre-treatment FFPE biopsy material revealed that high expression of the protein was associated with greater likelihood of CA19-9 decline. Paired pre- and on-treatment plasma samples were available for digital sequencing of ctDNA using the Guardant360 assay for 32 patients. Sequence variants likely originating from the tumor (based on allele frequency and absence in the above-mentioned germ-line variants) were found in 27 (84%) pre-treatment and 25 (78%) on-treatment samples. Most frequently mutated gene in pre-therapeutic plasma samples, in which circulating tumor fraction is > 0.4%, was KRAS (85%), followed by TP53 (60%), ATM (30%), and CDKN2A (15%). Relative change in allele frequency for those alleles that were present in pre- and on-treatment biopsies were positively correlated with differences in RECIST measurements. Conclusions: In summary, dual targeting of EGFR/MEK signaling shows antitumor activity in PDAC in a subset of patients, in particular those with high levels of E-cadherin expression, which is in agreement with our preclinical findings. Digital sequencing of ctDNA provides information about genomic alterations in the majority of patients and holds the potential of providing early information on response to therapy. Supported by CTEP and NIH R21 CA149939. Citation Format: Andrew H. Ko, Tanios Bekaii-Saab, Ryan Courtin, Olga K. Mirzoeva, Sharvina Ziyeh, Robin K. Kelley, Elizabeth Ditto, Anna Ong, Regina Linetskaya, Margaret Tempero, Alan P. Venook, Amirali Talasaz, Wolfgang Michael Korn. Next-generation sequence analysis of cell-free DNA in patients with chemotherapy-refractory advanced pancreatic adenocarcinoma (PDAC) treated with selumetinib (AZD6244) and erlotinib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5244. doi:10.1158/1538-7445.AM2015-5244

Abstract 986: Reverse-engineered, forward-simulation of MEK-dependent molecular networks reveal novel regulators of cell cycle and cancer cell survival

ABSTRACT Molecular networks governing responses to targeted therapies in cancer cells are complex dynamic systems that demonstrate non-intuitive behaviors. We applied a novel computational strategy to decipher causal relationships between signaling network components. Gene networks were constructed MEK from global gene expression profiles to model G1-S cell cycle arrest as a consequence of inhibition of MEK, a key component of the oncogenic RAF-MEK-ERK signal transduction pathway. Through forward-simulation of reverse-engineered networks candidate MEK-dependent regulators of the cell cycle were identified. The potential of the approach to infer causality is demonstrated by confirmed predictions of differential effects of two early-response transcription factors, IER2 and EGR1. Conventional linear regression analysis shows strong correlation of expression of both with MEK inhibition and cell cycle distribution, while only IER2 is predicted by our model and experimentally validated to have a causal effect on G1-S transition. Furthermore, we identify TRIB1 (tribbles homologue 1 drosophila) as a potent and novel cell cycle regulator. TRIB1 also mediates cross-talk to the NFkB pathway by enhancing p100 and IkBa-phosphorylation and degradation thus regulating cell survival and expression of cytokines crucial for tumor-stroma interaction. In agreement with this, knock-down of TRIB1 results in induction of apoptosis and sensitizes cells to cell killing induced by the death-receptor agonist TRAIL via upregulation of TRAIL receptor, DR5. Thus TRIB1 is a central regulator of cell cycle and survival and represents a potential therapeutic target. 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 986. doi:1538-7445.AM2012-986

Abstract A69: Subtype-specific targeting of RAS signal transduction in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA), the fourth-leading cause of cancer related death in the US, remains an incurable disease. Among the most promising targets for new treatments for PDA is the KRAS oncogene since mutations in this gene are found in about 90% of all cases. Because attempts to target the KRAS protein itself have failed so far, inhibiting pathways down-stream of KRAS in particular the MAPK and PI3K pathways, represent promising targets. We discovered that inhibition of MEK, a major effector of KRAS signaling, leads to feedback activation of the pro-survival PI3K/AKT pathway through an EGFR-mediated mechanism. In agreement with this finding, many PDA cell lines show synergistic growth inhibition and induction of apoptosis when treated with combinations of MEK and EGFR inhibitors or MEK with PI3K inhibitors. We profiled gene expression patterns in an extensive panel of human PDAC cell lines and identified two subtypes with epithelial and mesenchymal features, respectively. The vast majority of cell lines demonstrating synergism in response to combinations of MEK inhibitors (CI1040 or PD0325901) with EGFR inhibitors (erlotinib) were of epithelial sub-type. In contrast, none of the mesenchymal-type PDA lines showed synergistic effects with respect to apoptosis or cell growth inhibition. Predictor analysis using the cell line mRNA expression data revealed a 13-gene signature predictive of sensitivity to the drug combination. We hypothesize that these predictors will allow for the development of patient selection strategies for clinical application of combinations of MEK- and EGFR inhibitors in PDA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A69.