Barbara Lupo

The genomic landscape of response to EGFR blockade in colorectal cancer

Colorectal cancer is the third most common cancer worldwide, with 1.2 million patients diagnosed annually. In late-stage colorectal cancer, the most commonly used targeted therapies are the monoclonal antibodies cetuximab and panitumumab, which prevent epidermal growth factor receptor (EGFR) activation. Recent studies have identified alterations in KRAS and other genes as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in colorectal cancer and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in colorectal cancer on response to anti-EGFR antibody therapy, here we perform complete exome sequence and copy number analyses of 129 patient-derived tumour grafts and targeted genomic analyses of 55 patient tumours, all of which were KRAS wild-type. We analysed the response of tumours to anti-EGFR antibody blockade in tumour graft models and in clinical settings and functionally linked therapeutic responses to mutational data. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumours with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumour graft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluating response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and delineate new avenues for intervention in managing colorectal cancer.

IGF2 is an actionable target that identifies a distinct subpopulation of colorectal cancer patients with marginal response to anti-EGFR therapies

Colorectal cancers that display reduced sensitivity to EGFR inhibition and strong IGF2 overexpression can be effectively treated by dual EGFR/IGF2 blockade. Better together Inhibitors of the epidermal growth factor receptor (EGFR) are already used to treat colorectal cancer. Unfortunately, although many patients’ tumors respond to these drugs, most of these responses are only partial and result in a slowing of tumor growth rather than a regression of the cancer. Now, Zanella et al. used a combination of patient samples and mouse xenografts to determine the reasons for the incomplete response to treatment and how it can be overcome. In some cases, more effective treatment just required a more complete inhibition of EGFR. Many of the other cancers overexpressed insulin-like growth factor 2 (IGF2), and the authors discovered that combining inhibitors of EGFR and IGF was an effective way to overcome resistance in these tumors. Among patients with colorectal cancer who benefit from therapy targeted to the epidermal growth factor receptor (EGFR), stable disease (SD) occurs more frequently than massive regressions. Exploring the mechanisms of this incomplete sensitivity to devise more efficacious treatments will likely improve patients’ outcomes. We tested therapies tailored around hypothesis-generating molecular features in patient-derived xenografts (“xenopatients”), which originated from 125 independent samples that did not harbor established resistance-conferring mutations. Samples from xenopatients that responded to cetuximab, an anti-EGFR agent, with disease stabilization displayed high levels of EGFR family ligands and receptors, indicating high EGFR pathway activity. Five of 21 SD models (23.8%) characterized by particularly high expression of EGFR and EGFR family members regressed after intensified EGFR blockade by cetuximab and a small-molecule inhibitor. In addition, a subset of cases in which enhanced EGFR inhibition was unproductive (6 of 16, 37.5%) exhibited marked overexpression of insulin-like growth factor 2 (IGF2). Enrichment of IGF2 overexpressors among cases with SD was demonstrated in the entire xenopatient collection and was confirmed in patients by mining clinical gene expression data sets. In functional studies, IGF2 overproduction attenuated the efficacy of cetuximab. Conversely, interception of IGF2-dependent signaling in IGF2-overexpressing xenopatients potentiated the effects of cetuximab. The clinical implementation of IGF inhibitors awaits reliable predictors of response, but the results of this study suggest rational combination therapies for colorectal cancer and provide evidence for IGF2 as a biomarker of reduced tumor sensitivity to anti-EGFR therapy and a determinant of response to combined IGF2/EGFR targeting.

Rebound Effects Caused by Withdrawal of MET Kinase Inhibitor Are Quenched by a MET Therapeutic Antibody

MET oncogene amplification is emerging as a major mechanism of acquired resistance to EGFR-directed therapy in lung and colorectal cancers. Furthermore, MET amplification predicts responsiveness to MET inhibitors currently in clinical trials. Among the anti-MET drugs available, ATP-competitive small-molecule kinase inhibitors abrogate receptor autophosphorylation and downstream activation of ERK1/2 and AKT, resulting in cell-cycle arrest. However, this antiproliferative effect allows persistence of a pool of cancer cells that are quiescent but alive. Once the inhibition is removed, rebound activation of MET-driven cell proliferative pathways and tumor growth may occur, an adverse event observed frequently in clinical settings after drug discontinuation. Here we show that inhibitor withdrawal prompts receptor phosphorylation to levels higher than those displayed at steady-state and generates a rebound effect pushing quiescent cancer cells back into the cell cycle, both in vitro and in experimental tumor models in vivo Mechanistically, we found that inhibitor treatment blocks MET endocytosis, causing a local increase in the number of receptors at the plasma membrane. Upon inhibitor washout, the receptor is readily rephosphorylated. The initial phosphorylation is not only increased but also prolonged in duration due to downmodulation of a phosphatase-mediated MET-negative feedback loop, which accompanies receptor internalization. Notably, treatment with a MET therapeutic antibody that induces proteolytic cleavage of the receptor at the cell surface substantially prevents this rebound effect, providing a rationale to combine or alternate these mechanistically different types of MET-targeted therapy. Cancer Res; 76(17); 5019-29. ©2016 AACR.

Tankyrase inhibition impairs directional migration and invasion of lung cancer cells by affecting microtubule dynamics and polarity signals.

BackgroundTankyrases are poly(adenosine diphosphate)-ribose polymerases that contribute to biological processes as diverse as modulation of Wnt signaling, telomere maintenance, vesicle trafficking, and microtubule-dependent spindle pole assembly during mitosis. At interphase, polarized reshaping of the microtubule network fosters oriented cell migration. This is attained by association of adenomatous polyposis coli with the plus end of microtubules at the cortex of cell membrane protrusions and microtubule-based centrosome reorientation towards the migrating front.ResultsHere we report a new function for tankyrases, namely, regulation of directional cell locomotion. Using a panel of lung cancer cell lines as a model system, we found that abrogation of tankyrase activity by two different, structurally unrelated small-molecule inhibitors (one introduced and characterized here for the first time) or by RNA interference-based genetic silencing weakened cell migration, invasion, and directional movement induced by the motogenic cytokine hepatocyte growth factor. Mechanistically, the anti-invasive outcome of tankyrase inhibition could be ascribed to sequential deterioration of the distinct events that govern cell directional sensing. In particular, tankyrase blockade negatively impacted (1) microtubule dynamic instability; (2) adenomatous polyposis coli plasma membrane targeting; and (3) centrosome reorientation.ConclusionsCollectively, these findings uncover an unanticipated role for tankyrases in influencing at multiple levels the interphase dynamics of the microtubule network and the subcellular distribution of related polarity signals. These results encourage the further exploration of tankyrase inhibitors as therapeutic tools to oppose dissemination and metastasis of cancer cells.

Lenalidomide in the Treatment of Young Patients with Multiple Myeloma: From Induction to Consolidation/Maintenance Therapy

Multiple myeloma is the second most common hematologic malignancy. It accounts for 20,580 new cancer cases in the USA in 2009, including 11,680 cases in men, 8,900 cases in women, and 10,580 deaths overall. Although the disease remains still incurable, outcomes have improved substantially over recent years thanks to the use of high-dose therapy and the availability of novel agents, such as the immunomodulatory drugs thalidomide and lenalidomide, and the proteasome inhibitor bortezomib. Various trials have shown the advantages linked to the use of novel agents in the transplant and not-transplant settings. In particular, this paper will present an overview of the results achieved with lenalidomide-containing combinations in patients eligible for high-dose therapies, namely, young patients. The advantages obtained should always be outweighed with the toxicity profile associated with the regimen used. Therefore, here, we will also provide a description of the main adverse events associated with lenalidomide and its combination.

Abstract LB-293: Targeting the PI3K pathway to intercept cetuximab tolerance in metastatic colorectal cancer

In the precision medicine era, multi-parametric interrogation of tumors in cancer patients has enabled rationally refined clinical trials and considerably improved response rates. Nevertheless, incomplete mass obliteration and late relapse invariably emerge. This also relates to metastatic colorectal cancer (mCRC), as massive tumor regression upon anti-EGFR therapy with cetuximab is relatively infrequent. Clinical evidence suggests that tumor recurrence may be endorsed by a population of lingering cancer cells that survive therapy and fuel residual disease through reversible, non-mutational cues, thus installing a “poised” state of drug tolerance that anticipates tumor relapse. The mechanisms underlying this resilience, and the strategies to tackle it, remain to be explored. In this study, we capitalized on different exploratory resources, including a proprietary collection of patient-derived xenografts, as well as CRC cell lines and primary cultures, to explore the mechanisms of cetuximab tolerance. Biochemical characterization of cetuximab persisters evidenced a dichotomous output, with full MAPK neutralization but persistent PI3K/AKT activity, suggesting that PI3K is engaged as a competitor route. Consistently, concomitant EGFR/PI3K blockade specifically unleashed apoptosis in cell lines, as opposed to the cytostatic activity of cetuximab alone, and prevented colosphere growth recovery after drug washout. In vivo, evaluation of residual cellularity confirmed the superior efficacy of dual EGFR/PI3K deactivation. Hence, both the number and fitness of tolerant cells were thwarted by EGFR/PI3K interception. Integrative morphologic analysis, immunohistochemistry, gene-expression profiling and reporter assays in vitro put forward increased beta-catenin activity and Paneth cell-like reprogramming as distinctive traits of residual cells surviving cetuximab treatment. These features are likely related to cetuximab-induced biochemical rewiring, as relieve of ERK-mediated beta-catenin repression in conjunction with persistent PI3K-AKT activity may converge on Wnt-dependent Paneth cell-like differentiation. Importantly, PI3K inhibition dampened cetuximab-triggered Paneth cell specification, thus intercepting a putative mechanism of drug-tolerance. Future studies will extricate the relative contribution of PI3K, MAPK, and Wnt-dependent networks to sustaining the reservoir of persisters, and how Paneth-like traits may foster residual disease. Citation Format: Barbara Lupo, Paolo Gagliardi, Francesco Sassi, Eugenia Rosalinda Zanella, Francesca Cottino, Andrea Bertotti, Livio Trusolino. Targeting the PI3K pathway to intercept cetuximab tolerance in metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-293. doi:10.1158/1538-7445.AM2017-LB-293

Abstract A48: Tankyrase inhibitors impair directional cell migration of cancer cells by affecting microtubule dynamics and polarity signals

Tumor colonization of distant sites underlies the vast majority of cancer-related deaths worldwide, despite current therapeutic efforts. Unfortunately, the emergence of metastasis-competent clones characterizes the rapid outgrowth of highly aggressive tumors, such as lung cancer [1]. Therefore, dissecting the molecular mechanisms that drive metastatic dissemination is the obligatory groundwork to discover novel therapeutic targets. In an endeavor to identify potential anti-invasive compounds, we performed a large-scale drug screen coupled to cell-based strategies, including wound healing and Transwell assays, in a panel of non-small cell lung cancer (NSCLC) cell lines. This approach led to the identification of two small molecules, namely XAV939 and JNJ928, both displaying remarkable inhibitory effects on cell invasiveness, in the absence of overt cytotoxicity. Both compounds are known to inhibit tankyrases 1&2, two poly(ADP)-ribosylases that are gaining interest due to their involvement in cancer-related processes, ranging from wnt signaling to telomere maintenance and microtubule-based mitotic dynamics [2]. A scrutiny of polarity signaling pathways known to be involved in cell locomotion allowed us to allocate tankyrases within an accredited route governing directional cell migration that entails cdc42-dependent phosphorylation of GSK3β, the ensuing recruitment of APC to the plasma membrane, and the establishment of microtubule-dependent pulling forces that reorient the microtubule organizing center (MTOC) towards the leading edge [3]. Notably, all these spatially orchestrated events were compromised by tankyrase blockade, likely explaining the reduced chemotactic response of XAV939- and JNJ928-treated cells. Concurrently, tankyrase inhibition seemed to impinge on microtubule dynamics by stabilizing the microtubule network. Notably, dynamic instability of microtubules is needed for their “search and capture” function to generate an asymmetric microtubule array and facilitate cell movement [4]. Conceivably, suppression of dynamic instability by tankyrase blockade might prevent microtubules from probing outward, leading to reduced receptiveness of chemotactic inputs. Efforts are currently being placed to uncover the mechanistic details of such interconnections, which seem to adhere to the established notion that tankyrases bind to and regulate the activity of several microtubule-related proteins. Our results might add to the evolving landscape of tankyrases as drivers of many aspect of the transformed phenotype, holding promise for prospective – albeit still immature – employment of tankyrase inhibitors as anti-metastatic therapeutics in NSCLC. [1] D.X. Nguyen, P.D. Bos, J. Massague, Metastasis: from dissemination to organ-specific colonization, Nat Rev Cancer 9 (2009) 274-284. [2] J.L. Riffell, C.J. Lord, A. Ashworth, Tankyrase-targeted therapeutics: expanding opportunities in the PARP family, Nat Rev Drug Discov 11 (2012) 923-936. [3] S. Etienne-Manneville, Polarity proteins in migration and invasion, Oncogene 27 (2008) 6970-6980. [4] S. Etienne-Manneville, Microtubules in cell migration, Annu Rev Cell Dev Biol 29 (2013) 471-499. Citation Format: Barbara Lupo, Jorge Vialard, Andrea Bertotti, Letizia Lanzetti, Livio Trusolino. Tankyrase inhibitors impair directional cell migration of cancer cells by affecting microtubule dynamics and polarity signals. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A48.