Mark J. Axelrod

Synthetic Lethal Screening with Small-Molecule Inhibitors Provides a Pathway to Rational Combination Therapies for Melanoma

Recent data show that extracellular signals are transmitted through a network of proteins rather than hierarchical signaling pathways, suggesting that the inhibition of a single component of a canonical pathway is insufficient for the treatment of cancer. The biologic outcome of signaling through a network is inherently more robust and resistant to inhibition of a single network component. In this study, we conducted a functional chemical genetic screen to identify novel interactions between signaling inhibitors that would not be predicted on the basis of our current understanding of signaling networks. We screened over 300 drug combinations in nine melanoma cell lines and have identified pairs of compounds that show synergistic cytotoxicity. The synergistic cytotoxicities identified did not correlate with the known RAS and BRAF mutational status of the melanoma cell lines. Among the most robust results was synergy between sorafenib, a multikinase inhibitor with activity against RAF, and diclofenac, a nonsteroidal anti-inflammatory drug (NSAID). Drug substitution experiments using the NSAIDs celecoxib and ibuprofen or the MAP–ERK kinase inhibitor PD325901 and the RAF inhibitor RAF265 suggest that inhibition of COX and mitogen-activated protein kinase signaling are targets for the synergistic cytotoxicity of sorafenib and diclofenac. Cotreatment with sorafenib and diclofenac interrupts a positive feedback signaling loop involving extracellular signal–regulated kinase, cellular phospholipase A2, and COX. Genome-wide expression profiling shows synergy-specific downregulation of survival-related genes. This study has uncovered novel functional drug combinations and suggests that the underlying signaling networks that control responses to targeted agents can vary substantially, depending on unexplored components of the cell genotype. Mol Cancer Ther; 11(11); 2505–15. ©2012 AACR.

Combinatorial drug screening identifies synergistic co-targeting of Bruton’s tyrosine kinase and the proteasome in mantle cell lymphoma

Combinatorial drug screening identifies synergistic co-targeting of Bruton’s tyrosine kinase and the proteasome in mantle cell lymphoma

p70S6 kinase is a critical node that integrates HER-family and PI3 kinase signaling networks.

Therapies targeting oncogenic drivers rapidly induce compensatory adaptive responses that blunt drug effectiveness, contributing to therapeutic resistance. Adaptive responses are characteristic of robust cell signaling networks, and thus there is increasing interest in drug combinations that co-target the driver and the adaptive response. An alternative approach to co-inhibiting oncogenic and adaptive targets is to identify a critical node where the activities of these targets converge. Nodes of convergence between signaling modules represent potential therapeutic vulnerabilities because their inhibition could result in the collapse of the network, leading to enhanced cytotoxicity. In this report we demonstrate that p70S6 kinase (p70S6K) can function as a critical node linking HER-family and phosphoinositide-3-kinase (PI3K) pathway signaling. We used high-throughput combinatorial drug screening to identify adaptive survival responses to targeted therapies, and found that HER-family and PI3K represented compensatory signaling pathways. Co-targeting these pathways with drug combinations caused synergistic cytotoxicity in cases where inhibition of neither target was effective as a monotherapy. We utilized Reverse Phase Protein Arrays and determined that phosphorylation of ribosomal protein S6 was synergistically down-regulated upon HER-family and PI3K/mammalian target of rapamycin (mTOR) co-inhibition. Expression of constitutively active p70S6K protected against apoptosis induced by combined HER-family and PI3K/mTOR inhibition. Direct inhibition of p70S6K with small molecule inhibitors phenocopied HER-family and PI3K/mTOR co-inhibition. These data implicate p70S6K as a critical node in the HER-family/PI3K signaling network. The ability of direct inhibitors of p70S6K to phenocopy co-inhibition of two upstream signaling targets indicates that identification and targeting of critical nodes can overcome adaptive resistance to targeted therapies.

Systems Analysis of Adaptive Responses to MAP Kinase Pathway Blockade in BRAF Mutant Melanoma

Fifty percent of cutaneous melanomas are driven by activated BRAF V600E, but tumors treated with RAF inhibitors, even when they respond dramatically, rapidly adapt and develop resistance. Thus, there is a pressing need to identify the major mechanisms of intrinsic and adaptive resistance and develop drug combinations that target these resistance mechanisms. In a combinatorial drug screen on a panel of 12 treatment-naïve BRAF V600E mutant melanoma cell lines of varying levels of resistance to mitogen-activated protein kinase (MAPK) pathway inhibition, we identified the combination of PLX4720, a targeted inhibitor of mutated BRaf, and lapatinib, an inhibitor of the ErbB family of receptor tyrosine kinases, as synergistically cytotoxic in the subset of cell lines that displayed the most resistance to PLX4720. To identify potential mechanisms of resistance to PLX4720 treatment and synergy with lapatinib treatment, we performed a multi-platform functional genomics analysis to profile the genome as well as the transcriptional and proteomic responses of these cell lines to treatment with PLX4720. We found modest levels of resistance correlated with the zygosity of the BRAF V600E allele and receptor tyrosine kinase (RTK) mutational status. Layered over base-line resistance was substantial upregulation of many ErbB pathway genes in response to BRaf inhibition, thus generating the vulnerability to combination with lapatinib. The transcriptional responses of ErbB pathway genes are associated with a number of transcription factors, including ETS2 and its associated cofactors that represent a convergent regulatory mechanism conferring synergistic drug susceptibility in the context of diverse mutational landscapes.

Abstract 5633: Co-targeting the IGF1R pathway and compensatory signaling enhances cytotoxicity in head and neck cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The 5-year survival rate for patients with cancer of the Head and Neck (HNC) has not significantly improved over the past decade. Clearly, new therapeutic targets and strategies for employing existing targeted and cytotoxic therapies are needed. Studies have shown both expression and phosphorylation of the insulin-like growth factor 1 receptor (IGF1R) in both head and neck cancer cell lines and patient tumor samples. We found that small molecule inhibitors targeting IGF1R, when used as a single agent, caused varying degrees of cytotoxicity in HNC cell lines. In order to determine whether the variable biological response to IGF1R inhibition was caused by differences in the effects of the IGF1R inhibitors on the cell signaling networks of the HNC cell lines, we performed reverse phase proteomic array analysis on a panel of HNC cell lines treated with IGF1R inhibitors. Preliminary results suggest that treatment with IGF1R inhibitors caused increased expression and/or phosphorylation of a number of proteins in the array. We hypothesize that these alterations represent compensatory signaling pathways that provide resistance to cytotoxicity upon treatment with IGF1R inhibitors. In order to identify possible mechanisms of compensation for the loss of IGF1R pathway signaling, we screened a panel of small molecule inhibitors of the IGF1R/PI3K/AKT signaling pathway against a panel of inhibitors of proteins important in HNC cell signaling Preliminary results indicate that the inhibition of epigenetic modifying proteins as well as members of canonical cell signaling pathways in combination with inhibition of IGF1R signaling leads to a synergistic increase in cytotoxicity. This suggests that compensatory mechanisms exist in HNC that serve to blunt the cytotoxic effect of inhibition of IGF1R. Co-targeting IGF1R and members of these compensatory pathways may be a viable therapeutic strategy. Citation Format: Mark J. Axelrod, Daniel Gioeli, Elizabeth R. Sharlow, Mark C. Conaway, Rolando E. Mendez, Ashraf Khalil, Linnea Taniguchi, Emmanual F. Petricoin, Stephanie Leimgruber, Michael J. Weber, Mark J. Jameson. Co-targeting the IGF1R pathway and compensatory signaling enhances cytotoxicity in head and neck cancer. [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 5633. doi:10.1158/1538-7445.AM2013-5633

Synergistic apoptosis in head and neck squamous cell carcinoma cells by co-inhibition of insulin-like growth factor-1 receptor signaling and compensatory signaling pathways

In head and neck squamous cell carcinoma (HNSCC), resistance to single‐agent targeted therapy may be overcome by co‐targeting of compensatory signaling pathways.

Abstract B40: Synergistic cytotoxicity of ibrutinib and the BCL2 antagonist ABT-199 in mantle cell lymphoma and chronic lymphocytic leukemia: Molecular analysis reveals mechanisms of target interactions.

Bruton tyrosine kinase (BTK) is critical to both normal B-cell development and the pathogenesis of B-cell malignancies. Ibrutinib is a recently FDA-approved small molecule irreversible inhibitor of BTK, but not all patients respond and complete responses are infrequent with single agent ibrutinib. We hypothesize that additional agents used in combination could enhance the breadth, depth and duration of responses. We previously reported that the BCL2 inhibitor, ABT-199, and the proteasome inhibitor, carfilzomib, were highly synergistic with ibrutinib in MCL cell lines (Axelrod M et al, Leukemia 2014). We sought to confirm these findings in MCL and CLL patient samples and to determine the mechanisms of synergy. Peripheral blood buffy coat samples from patients with circulating tumor cells were exposed to ibrutinib, ABT-199, carfilzomib and the combinations of ibrutinib and ABT-199 and ibrutinib and carfilzomib at pharmacologically-achievable doses for 72 hours. Apoptosis was assessed using PARP cleavage by FACS analysis of CD3-, CD5+, CD19+ cells representing the neoplastic clones. The combination of Ibrutinib and ABT-199 displayed synergistic cytotoxicity (combo: 23%, ibrutinib: 3.8%, ABT-199: 3.0%). Ibrutinib plus carfilzomib also substantially induced apoptosis compared to each single agent alone (combo: 5.5%, Ibrutinib 3.8%, carfilzomib 1.7%) though to a less degree than the ABT-199 combination. The normal B-cell population (CD3-, CD5-, CD19+) in these samples was too small for analysis, thus normal T-cells (CD3+, CD5+, CD19-) from the same patients were used to identify the effects on normal lymphocytes. Minimal apoptosis was seen in normal T-cells with the single agents or the combinations. In a cohort of CLL cells from 9 patients, 5 displayed synergistic cytotoxicity and 4 did not, indicating substantial patient heterogeneity in response to the combination, presumably due to variations in genetic landscape. No increased apoptosis was seen in two tested peripheral blood lymphocyte (CD3-, CD5-, CD19+) populations from healthy donors. Gene expression profiling with Illumina Bead Chip array was used to evaluate the mechanisms of synergy with ABT-199 plus ibrutinib after 6 hours of drug exposure. The MCL cell line JVM2 was exposed to pharmacologically-achievable doses of ibrutinib, ABT-199 and combinations of each dose. Ibrutinib alone induced transcriptional change whereas ABT-199 did little to change gene expression. The combination induced both potentiative transcriptional changes (changes present in isolation and enhanced by the combination) and emergent transcriptional changes (changes only seen with the combination, unchanged by each single agent). Protein-protein interaction networks generated using the drug targets (BTK and BCL2) and emergent genes as input to STRING revealed activation of apoptosis via p53 and BIM as mechanisms of synergy. In conclusion, Ibrutinib and ABT-199 induce synergistic apoptosis in MCL cell lines and leukemic patient samples. The combination also induced apoptosis in some, but not all, CLL patient samples. No apoptosis was seen with either drug or the combination in normal T-cells from patients, suggesting little off-target effect. Emergent changes were seen when combining ABT-199 with ibrutinib in MCL cell lines. These changes suggest activation of p53 and BIM as potential mechanisms of synergy. A clinical trial with ABT-199 and ibrutinib is planned. Citation Format: Craig A. Portell, Mark J. Axelrod, Laura Kyle Brett, Vicki L. Gordon, Brian Capaldo, Jeffrey Xing, Stefan Bekiranov, Michael E. Williams, Michael J. Weber. Synergistic cytotoxicity of ibrutinib and the BCL2 antagonist ABT-199 in mantle cell lymphoma and chronic lymphocytic leukemia: Molecular analysis reveals mechanisms of target interactions. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B40.

Abstract 902: XRCC1 induction after cisplatin treatment in head and neck squamous carcinoma cell lines: Evaluation using nanoimmunoassay

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The objective of this study was to use nanoimmunoassay (NIA) to quantify XRCC1 levels and correlate these with response to platinum-based chemotherapeutic agents in HNSCC cell lines. NIA is a developing technique that uses nanogram-scale samples to quantify specific proteins by immunoassay after separation by isoelectric focusing. Detection is more sensitive and quantification more accurate than with standard immunoblot techniques. XRCC1 is a base-excision repair protein that has been identified as a possible mediator of resistance to platinum-based chemotherapeutic agents, but its role and predictive utility have undergone limited investigation in head and neck squamous cell carcinoma (HNSCC). Detection of XRCC1 by NIA was first optimized. In lysates of HNSCC cell lines, XRCC1 was consistently identified at a pI of 5.45 ± 0.1. Peak intensity was improved by the addition of 7 M urea to the specimen prior to loading. In order to correct for sample and loading variability, and detection of a simultaneous internal loading control was also optimized. Under conditions that were optimal for detection of XRCC1, thioredoxin as consistently identified at a pI of 4.90 ± 0.1. Using 9 HNSCC cell lines, XRCC1 levels were evaluated using whole cell lysates in the untreated (basal) state as well as 24 h after treatment with cisplatin or vehicle. NIA was performed on 200 ng of whole cell lysate and XRCC1 and thioredoxin peak areas were quantified using the associated software. Proliferation assays were performed using alamarBlue to determine the IC50 for cisplatin in each cell line. Basal XRCC1 expression levels did not correlate with sensitivity to cisplatin. However, XRCC1 levels were noted to be significantly altered after treatment. When XRCC1 levels were normalized to the thioredoxin loading control, the ratio of treated to basal XRCC1 was correlated to the IC50 for cisplatin across all cell lines (R2=0.428). In cell lines identified as resistant to cisplatin, XRCC1 levels increased upon treatment, whereas in sensitive cell lines, levels remained unchanged. These NIA findings were confirmed using XRCC1 immunoblot with beta-actin as a loading control; XRCC1 levels obtained using this approach also correlated with the IC50 for cisplatin (R2=0.518). While timing and methodology need to be further elucidated, the change in XRCC1 protein level in response to a cisplatin challenge has the potential to be used as a biomolecular predictor of sensitivity that could direct treatment modality selection early in the course of therapy for HNSCC patients. The quantities of protein used allow for potential evaluation of fine-needle aspirates in patients undergoing treatment. Citation Format: Stephen S. Schoeff, Dane M. Barrett, James Teng, Ashraf Khalil, Matthew A. Hubbard, Anne K. Maxwell, Amir Allak, Rolando E. Mendez, Mark Axelrod, Mark J. Jameson. XRCC1 induction after cisplatin treatment in head and neck squamous carcinoma cell lines: Evaluation using nanoimmunoassay. [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 902. doi:10.1158/1538-7445.AM2014-902

Abstract 695: p70S6 kinase is a critical node that integrates HER-family and PI3 kinase signaling

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Targeted cancer therapies often induce compensatory adaptive responses that blunt drug effectiveness, resulting in therapeutic resistance - intrinsic or acquired. Adaptive responses are characteristic of the complex nature of the cancer cell signaling network. Within this network, nodes of convergence between signaling modules are critical mediators of adaptive responses to targeted drugs. The critical nodes in an oncogenically-activated signaling network represent potential therapeutic vulnerabilities because their inhibition could result in collapse of the network and hence enhanced cytotoxicity. We have previously used high-throughput combinatorial drug screening to empirically identify adaptive survival responses to targeted therapies. We found that HER-family and PI3K represented compensatory signaling pathways, and combination therapy caused synergistic cytotoxicity in cases where inhibition of neither target was effective as a monotherapy. RPPA analysis identified ribosomal protein S6 as being synergistically down-regulated upon HER-family and PI3K co-inhibition. Expression of a constitutively active construct of the upstream activator of S6, p70S6K, was protective against apoptosis induced by combined HER-family and PI3K inhibition. Direct inhibition of p70S6K using small molecule inhibitors phenocopied the growth inhibition and apoptosis caused by HER-family and PI3K inhibition. These data implicate p70S6K as a critical node and druggable target in the HER-family / PI3K signaling network. The ability of direct inhibitors of p70S6K to phenocopy the co-inhibition of upstream signaling indicates that identification and targeting of critical nodes may be a way to overcome adaptive resistance to targeted therapies. Citation Format: Mark J. Axelrod, Rolando E. Mendez, Daniel G. Gioeli, Mark J. Jameson, Michael J. Weber. p70S6 kinase is a critical node that integrates HER-family and PI3 kinase signaling. [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 695. doi:10.1158/1538-7445.AM2014-695

Abstract 4166: Integrated molecular profiling of melanoma cell lines reveals genotype-drug phenotype associations giving insight on development of combination therapies

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Integrated molecular profiling of melanoma cell lines reveals genotype-drug phenotype associations giving insight on development of combination therapies Adaptive survival responses to targeted therapy reduce therapeutic benefit, and thus there is a need to identify drug combinations that co-target both oncogenic drivers and critical adaptive responses. We screened a panel of twelve V600E BRAF melanoma cell lines with a library of targeted drugs, and identified the combination of lapatinib (a HER family inhibitor) plus PLX4720 (a RAF inhibitor) as synergistically cytotoxic in the six lines that displayed complete or partial resistance to PLX4720 alone. To understand the mechanism(s) of drug resistance and synergy, and to identify potential biomarkers for utilization of this combination, we have performed an extensive molecular analysis of the cell lines and their responses to treatment with the drugs, singly and in combination. Alamar blue was used to calculate the cytotoxic effects of the drugs, and the cells were profiled by gene expression microarrays and reverse phase protein microarrays, before and after drug treatment. DNA methylation microarrays, and exome sequencing were used to identify the epigenetic and genetic context of the lines. Unsupervised hierarchical clustering analysis of the cytotoxic effects of the single drug and combinatorial treatments revealed four well defined groups: synergistic to combination treatment, PLX4720 sensitive, lapatinib resistant, and insensitive to either drug. The cell lines were analyzed using principal component analysis on the basal gene expression profiles. This analysis revealed four major clusters whose membership was identical to the clusters observed in the cytotoxicity analysis, indicating a strong genotype-drug phenotype relationship (i.e. cell lines that showed similar basal transcriptional programs also demonstrated similar responses to treatment. Differential gene expression analysis was performed between the synergistic group and each of the other groups. The most prominent gene expression differences were between the synergistic and PLX4720 sensitive lines, which correlated with differences in MITF expression and transcriptional output. Changes in gene expression and phosphoproteome levels in response to PLX4720 and lapatinib treatment between the four groups were used to identify genes and pathways associated with drug resistance and synergy. Genes involved in apoptosis and cell cycle regulation were found that could play a role in conferring resistance and synergistic response to PLX4720 and lapatinib treatment. These differing patterns of adaptive responses provide insights into molecular mechanisms of drug resistance and synergy and a pathway to rational construction of drug combinations. Citation Format: Brian J. Capaldo, Devin G. Roller, Mark Axelrod, Alex Koeppel, Michael J. Weber, Aaron Mackey, Dan Gioeli, Stefan Bekiranov. Integrated molecular profiling of melanoma cell lines reveals genotype-drug phenotype associations giving insight on development of combination therapies. [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 4166. doi:10.1158/1538-7445.AM2014-4166