Publications by authors named "Livio Trusolino"

97 Publications

Dynamic and adaptive cancer stem cell population admixture in colorectal neoplasia.

Cell Stem Cell 2022 Aug;29(8):1213-1228.e8

Wellcome Centre Human Genetics, Roosevelt Drive, University of Oxford, Oxford, UK; Department of Pathology and Molecular Pathology, University and University Hospital Zürich, Rämistrasse 100, 8006 Zürich, Switzerland. Electronic address:

Intestinal homeostasis is underpinned by LGR5+ve crypt-base columnar stem cells (CBCs), but following injury, dedifferentiation results in the emergence of LGR5-ve regenerative stem cell populations (RSCs), characterized by fetal transcriptional profiles. Neoplasia hijacks regenerative signaling, so we assessed the distribution of CBCs and RSCs in mouse and human intestinal tumors. Using combined molecular-morphological analysis, we demonstrate variable expression of stem cell markers across a range of lesions. The degree of CBC-RSC admixture was associated with both epithelial mutation and microenvironmental signaling disruption and could be mapped across disease molecular subtypes. The CBC-RSC equilibrium was adaptive, with a dynamic response to acute selective pressure, and adaptability was associated with chemoresistance. We propose a fitness landscape model where individual tumors have equilibrated stem cell population distributions along a CBC-RSC phenotypic axis. Cellular plasticity is represented by position shift along this axis and is influenced by cell-intrinsic, extrinsic, and therapeutic selective pressures.
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http://dx.doi.org/10.1016/j.stem.2022.07.008DOI Listing
August 2022

TRF2 cooperates with CTCF for controlling the oncomiR-193b-3p in colorectal cancer.

Cancer Lett 2022 05 28;533:215607. Epub 2022 Feb 28.

Oncogenomic and Epigenetic Unit, IRCCS - Regina Elena National Cancer Institute, via Elio Chianesi 53, Rome, 00144, Italy. Electronic address:

The Telomeric Repeat binding Factor 2 (TRF2), a key protein involved in telomere integrity, is over-expressed in several human cancers and promotes tumor formation and progression. Recently, TRF2 has been also found outside telomeres where it can affect gene expression. Here we provide evidence that TRF2 is able to modulate the expression of microRNAs (miRNAs), small non-coding RNAs altered in human tumors. Among the miRNAs regulated by TRF2, we focused on miR-193b-3p, an oncomiRNA that positively correlates with TRF2 expression in human colorectal cancer patients from The Cancer Genome Atlas dataset. At the mechanistic level, the control of miR-193b-3p expression requires the cooperative activity between TRF2 and the chromatin organization factor CTCF. We found that CTCF physically interacts with TRF2, thus driving the proper positioning of TRF2 on a binding site located upstream the miR-193b-3p host-gene. The binding of TRF2 on the identified region is necessary for promoting the expression of miR-193b3p which, in turn, inhibits the translation of the onco-suppressive methyltransferase SUV39H1 and promotes tumor cell proliferation. The translational relevance of the oncogenic properties of miR-193b-3p was confirmed in patients, in whom the association between TRF2 and miR-193b-3p has a prognostic value.
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http://dx.doi.org/10.1016/j.canlet.2022.215607DOI Listing
May 2022

Effective drug combinations in breast, colon and pancreatic cancer cells.

Nature 2022 03 23;603(7899):166-173. Epub 2022 Feb 23.

Wellcome Sanger Institute, Cambridge, UK.

Combinations of anti-cancer drugs can overcome resistance and provide new treatments. The number of possible drug combinations vastly exceeds what could be tested clinically. Efforts to systematically identify active combinations and the tissues and molecular contexts in which they are most effective could accelerate the development of combination treatments. Here we evaluate the potency and efficacy of 2,025 clinically relevant two-drug combinations, generating a dataset encompassing 125 molecularly characterized breast, colorectal and pancreatic cancer cell lines. We show that synergy between drugs is rare and highly context-dependent, and that combinations of targeted agents are most likely to be synergistic. We incorporate multi-omic molecular features to identify combination biomarkers and specify synergistic drug combinations and their active contexts, including in basal-like breast cancer, and microsatellite-stable or KRAS-mutant colon cancer. Our results show that irinotecan and CHEK1 inhibition have synergistic effects in microsatellite-stable or KRAS-TP53 double-mutant colon cancer cells, leading to apoptosis and suppression of tumour xenograft growth. This study identifies clinically relevant effective drug combinations in distinct molecular subpopulations and is a resource to guide rational efforts to develop combinatorial drug treatments.
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http://dx.doi.org/10.1038/s41586-022-04437-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8891012PMC
March 2022

Chromatin Velocity reveals epigenetic dynamics by single-cell profiling of heterochromatin and euchromatin.

Nat Biotechnol 2022 02 11;40(2):235-244. Epub 2021 Oct 11.

Functional Genomics of Cancer Unit, Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milano, Italy.

Recent efforts have succeeded in surveying open chromatin at the single-cell level, but high-throughput, single-cell assessment of heterochromatin and its underlying genomic determinants remains challenging. We engineered a hybrid transposase including the chromodomain (CD) of the heterochromatin protein-1α (HP-1α), which is involved in heterochromatin assembly and maintenance through its binding to trimethylation of the lysine 9 on histone 3 (H3K9me3), and developed a single-cell method, single-cell genome and epigenome by transposases sequencing (scGET-seq), that, unlike single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq), comprehensively probes both open and closed chromatin and concomitantly records the underlying genomic sequences. We tested scGET-seq in cancer-derived organoids and human-derived xenograft (PDX) models and identified genetic events and plasticity-driven mechanisms contributing to cancer drug resistance. Next, building upon the differential enrichment of closed and open chromatin, we devised a method, Chromatin Velocity, that identifies the trajectories of epigenetic modifications at the single-cell level. Chromatin Velocity uncovered paths of epigenetic reorganization during stem cell reprogramming and identified key transcription factors driving these developmental processes. scGET-seq reveals the dynamics of genomic and epigenetic landscapes underlying any cellular processes.
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http://dx.doi.org/10.1038/s41587-021-01031-1DOI Listing
February 2022

Molecular Subtyping Combined with Biological Pathway Analyses to Study Regorafenib Response in Clinically Relevant Mouse Models of Colorectal Cancer.

Clin Cancer Res 2021 11 23;27(21):5979-5992. Epub 2021 Aug 23.

Department of Physiology and Medical Physics, Precision Cancer Medicine Group, Royal College of Surgeons in Ireland, Dublin, Ireland.

Purpose: Regorafenib (REG) is approved for the treatment of metastatic colorectal cancer, but has modest survival benefit and associated toxicities. Robust predictive/early response biomarkers to aid patient stratification are outstanding. We have exploited biological pathway analyses in a patient-derived xenograft (PDX) trial to study REG response mechanisms and elucidate putative biomarkers.

Experimental Design: Molecularly subtyped PDXs were annotated for REG response. Subtyping was based on gene expression (CMS, consensus molecular subtype) and copy-number alteration (CNA). Baseline tumor vascularization, apoptosis, and proliferation signatures were studied to identify predictive biomarkers within subtypes. Phospho-proteomic analysis was used to identify novel classifiers. Supervised RNA sequencing analysis was performed on PDXs that progressed, or did not progress, following REG treatment.

Results: Improved REG response was observed in CMS4, although intra-subtype response was variable. Tumor vascularity did not correlate with outcome. In CMS4 tumors, reduced proliferation and higher sensitivity to apoptosis at baseline correlated with response. Reverse phase protein array (RPPA) analysis revealed 4 phospho-proteomic clusters, one of which was enriched with non-progressor models. A classification decision tree trained on RPPA- and CMS-based assignments discriminated non-progressors from progressors with 92% overall accuracy (97% sensitivity, 67% specificity). Supervised RNA sequencing revealed that higher basal expression is associated with REG resistance.

Conclusions: Subtype classification systems represent canonical "" (starting points) to support REG biomarker identification, and provide a platform to identify resistance mechanisms and novel contexts of vulnerability. Incorporating functional characterization of biological systems may optimize the biomarker identification process for multitargeted kinase inhibitors.
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http://dx.doi.org/10.1158/1078-0432.CCR-21-0818DOI Listing
November 2021

Synthetic Lethality Screening Highlights Colorectal Cancer Vulnerability to Concomitant Blockade of NEDD8 and EGFR Pathways.

Cancers (Basel) 2021 Jul 28;13(15). Epub 2021 Jul 28.

Candiolo Cancer Institute, FPO-IRCCS, 10060 Candiolo, Italy.

Colorectal cancer (CRC) is a heterogeneous disease showing significant variability in clinical aggressiveness. Primary and acquired resistance limits the efficacy of available treatments, and identification of effective drug combinations is needed to further improve patients' outcomes. We previously found that the NEDD8-activating enzyme inhibitor pevonedistat induced tumor stabilization in preclinical models of poorly differentiated, clinically aggressive CRC resistant to available therapies. To identify drugs that can be effectively combined with pevonedistat, we performed a "drop-out" loss-of-function synthetic lethality screening with an shRNA library covering 200 drug-target genes in four different CRC cell lines. Multiple screening hits were found to be involved in the EGFR signaling pathway, suggesting that, rather than inhibition of a specific gene, interference with the EGFR pathway at any level could be effectively leveraged for combination therapies based on pevonedistat. Exploiting both -mutant and wild-type CRC models, we validated the therapeutic relevance of our findings by showing that combined blockade of NEDD8 and EGFR pathways led to increased growth arrest and apoptosis both in vitro and in vivo. Pathway modulation analysis showed that compensatory feedback loops induced by single treatments were blunted by the combinations. These results unveil possible therapeutic opportunities in specific CRC clinical settings.
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http://dx.doi.org/10.3390/cancers13153805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8345131PMC
July 2021

The gRASs Is Greener: Potential New Therapies in Lung Cancer with Acquired Resistance to KRAS Inhibitors.

Cancer Discov 2021 08;11(8):1874-1876

Department of Oncology, University of Torino, Candiolo, Torino, Italy.

Inhibitors of KRAS that bind the target in its inactive conformation and lock it in off-mode have shown early signs of clinical activity in patients with -mutant lung cancer, but responses tend to be short-lived and invariably prelude the development of acquired resistance through largely unexplored mechanisms. A new study describes the emergence of RAS-MAPK heterogeneous subclonal alterations in a patient relapsed on a KRAS inactive-state inhibitor and identifies a novel KRAS-resistant variant that is druggable by a next-generation compound capable of associating with KRAS in its active configuration..
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http://dx.doi.org/10.1158/2159-8290.CD-21-0609DOI Listing
August 2021

The heme synthesis-export system regulates the tricarboxylic acid cycle flux and oxidative phosphorylation.

Cell Rep 2021 06;35(11):109252

Molecular Biotechnology Center (MBC), Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy. Electronic address:

Heme is an iron-containing porphyrin of vital importance for cell energetic metabolism. High rates of heme synthesis are commonly observed in proliferating cells. Moreover, the cell-surface heme exporter feline leukemia virus subgroup C receptor 1a (FLVCR1a) is overexpressed in several tumor types. However, the reasons why heme synthesis and export are enhanced in highly proliferating cells remain unknown. Here, we illustrate a functional axis between heme synthesis and heme export: heme efflux through the plasma membrane sustains heme synthesis, and implementation of the two processes down-modulates the tricarboxylic acid (TCA) cycle flux and oxidative phosphorylation. Conversely, inhibition of heme export reduces heme synthesis and promotes the TCA cycle fueling and flux as well as oxidative phosphorylation. These data indicate that the heme synthesis-export system modulates the TCA cycle and oxidative metabolism and provide a mechanistic basis for the observation that both processes are enhanced in cells with high-energy demand.
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http://dx.doi.org/10.1016/j.celrep.2021.109252DOI Listing
June 2021

Precision oncology in metastatic colorectal cancer - from biology to medicine.

Nat Rev Clin Oncol 2021 08 16;18(8):506-525. Epub 2021 Apr 16.

Department of Oncology, University of Torino, Candiolo, Italy.

Remarkable progress has been made in the development of biomarker-driven targeted therapies for patients with multiple cancer types, including melanoma, breast and lung tumours, although precision oncology for patients with colorectal cancer (CRC) continues to lag behind. Nonetheless, the availability of patient-derived CRC models coupled with in vitro and in vivo pharmacological and functional analyses over the past decade has finally led to advances in the field. Gene-specific alterations are not the only determinants that can successfully direct the use of targeted therapy. Indeed, successful inhibition of BRAF or KRAS in metastatic CRCs driven by activating mutations in these genes requires combinations of drugs that inhibit the mutant protein while at the same time restraining adaptive resistance via CRC-specific EGFR-mediated feedback loops. The emerging paradigm is, therefore, that the intrinsic biology of CRC cells must be considered alongside the molecular profiles of individual tumours in order to successfully personalize treatment. In this Review, we outline how preclinical studies based on patient-derived models have informed the design of practice-changing clinical trials. The integration of these experiences into a common framework will reshape the future design of biology-informed clinical trials in this field.
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http://dx.doi.org/10.1038/s41571-021-00495-zDOI Listing
August 2021

Rational Treatment of Metastatic Colorectal Cancer: A Reverse Tale of Men, Mice, and Culture Dishes.

Cancer Discov 2021 07 5;11(7):1644-1660. Epub 2021 Apr 5.

Department of Oncology, University of Torino, Candiolo, Torino, Italy.

Stratification of colorectal cancer into subgroups with different response to therapy was initially guided by descriptive associations between specific biomarkers and treatment outcome. Recently, preclinical models based on propagatable patient-derived tumor samples have yielded an improved understanding of disease biology, which has facilitated the functional validation of correlative information and the discovery of novel response determinants, therapeutic targets, and mechanisms of tumor adaptation and drug resistance. We review the contribution of patient-derived models to advancing colorectal cancer characterization, discuss their influence on clinical decision-making, and highlight emerging challenges in the interpretation and clinical transferability of results obtainable with such approaches. SIGNIFICANCE: Association studies in patients with colorectal cancer have led to the identification of response biomarkers, some of which have been implemented as companion diagnostics for therapeutic decisions. By enabling biological investigation in a clinically relevant experimental context, patient-derived colorectal cancer models have proved useful to examine the causal role of such biomarkers in dictating drug sensitivity and are providing fresh knowledge on new actionable targets, dynamics of tumor evolution and adaptation, and mechanisms of drug resistance.
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http://dx.doi.org/10.1158/2159-8290.CD-20-1531DOI Listing
July 2021

Quantifying single-cell ERK dynamics in colorectal cancer organoids reveals EGFR as an amplifier of oncogenic MAPK pathway signalling.

Nat Cell Biol 2021 04 1;23(4):377-390. Epub 2021 Apr 1.

Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands.

Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC.
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http://dx.doi.org/10.1038/s41556-021-00654-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610573PMC
April 2021

Conservation of copy number profiles during engraftment and passaging of patient-derived cancer xenografts.

Nat Genet 2021 01 7;53(1):86-99. Epub 2021 Jan 7.

Center for Cancer Biology, VIB, Leuven, Belgium.

Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution during PDX engraftment and propagation, affecting the accuracy of PDX modeling of human cancer. Here, we exhaustively analyze copy number alterations (CNAs) in 1,451 PDX and matched patient tumor (PT) samples from 509 PDX models. CNA inferences based on DNA sequencing and microarray data displayed substantially higher resolution and dynamic range than gene expression-based inferences, and they also showed strong CNA conservation from PTs through late-passage PDXs. CNA recurrence analysis of 130 colorectal and breast PT/PDX-early/PDX-late trios confirmed high-resolution CNA retention. We observed no significant enrichment of cancer-related genes in PDX-specific CNAs across models. Moreover, CNA differences between patient and PDX tumors were comparable to variations in multiregion samples within patients. Our study demonstrates the lack of systematic copy number evolution driven by the PDX mouse host.
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http://dx.doi.org/10.1038/s41588-020-00750-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7808565PMC
January 2021

Implementing Systems Modelling and Molecular Imaging to Predict the Efficacy of BCL-2 Inhibition in Colorectal Cancer Patient-Derived Xenograft Models.

Cancers (Basel) 2020 Oct 14;12(10). Epub 2020 Oct 14.

Department of Physiology and Medical Physics, Centre for Systems Medicine, Royal College of Surgeons in Ireland, D02 YN77 Dublin, Ireland.

Resistance to chemotherapy often results from dysfunctional apoptosis, however multiple proteins with overlapping functions regulate this pathway. We sought to determine whether an extensively validated, deterministic apoptosis systems model, 'DR_MOMP', could be used as a stratification tool for the apoptosis sensitiser and BCL-2 antagonist, ABT-199 in patient-derived xenograft (PDX) models of colorectal cancer (CRC). Through quantitative profiling of BCL-2 family proteins, we identified two PDX models which were predicted by DR_MOMP to be sufficiently sensitive to 5-fluorouracil (5-FU)-based chemotherapy (CRC0344), or less responsive to chemotherapy but sensitised by ABT-199 (CRC0076). Treatment with ABT-199 significantly improved responses of CRC0076 PDXs to 5-FU-based chemotherapy, but showed no sensitisation in CRC0344 PDXs, as predicted from systems modelling. F-Fluorodeoxyglucose positron emission tomography/computed tomography (F-FDG-PET/CT) scans were performed to investigate possible early biomarkers of response. In CRC0076, a significant post-treatment decrease in mean standard uptake value was indeed evident only in the combination treatment group. Radiomic CT feature analysis of pre-treatment images in CRC0076 and CRC0344 PDXs identified features which could phenotypically discriminate between models, but were not predictive of treatment responses. Collectively our data indicate that systems modelling may identify metastatic (m)CRC patients benefitting from ABT-199, and that F-FDG-PET could independently support such predictions.
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http://dx.doi.org/10.3390/cancers12102978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7602510PMC
October 2020

Intratumoral Transcriptome Heterogeneity Is Associated With Patient Prognosis and Sidedness in Patients With Colorectal Cancer Treated With Anti-EGFR Therapy From the CO.20 Trial.

JCO Precis Oncol 2020 29;4. Epub 2020 Sep 29.

Division of Molecular Pathology, The Institute of Cancer Research, London, United Kingdom.

Purpose: Metastatic colorectal cancers (mCRCs) assigned to the transit-amplifying (TA) CRCAssigner subtype are more sensitive to anti-epidermal growth factor receptor (EGFR) therapy. We evaluated the association between the intratumoral presence of TA signature (TA-high/TA-low, dubbed as TA-ness classification) and outcomes in CRCs treated with anti-EGFR therapy.

Patients And Methods: The TA-ness classes were defined in a discovery cohort (n = 84) and independently validated in a clinical trial (CO.20; cetuximab monotherapy arm; n = 121) and other samples using an established NanoString-based gene expression assay. Progression-free survival (PFS), overall survival (OS), and disease control rate (DCR) according to TA-ness classification were assessed by univariate and multivariate analyses.

Results: The TA-ness was measured in 772 samples from 712 patients. Patients (treated with anti-EGFR therapy) with TA-high tumors had significantly longer PFS (discovery hazard ratio [HR], 0.40; 95% CI, 0.25 to 0.64; < .001; validation HR, 0.65; 95% CI, 0.45 to 0.93; = .018), longer OS (discovery HR, 0.48; 95% CI, 0.29 to 0.78; = .003; validation HR, 0.67; 95% CI, 0.46 to 0.98; = .04), and higher DCR (discovery odds ratio [OR]; 14.8; 95% CI, 4.30 to 59.54; < .001; validation OR, 4.35; 95% CI, 2.00 to 9.09; < .001). TA-ness classification and its association with anti-EGFR therapy outcomes were further confirmed using publicly available data (n = 80) from metastatic samples (PFS < .001) and patient-derived xenografts ( = .042). In an exploratory analysis of 55 patients with wild-type and left-sided tumors, TA-high class was significantly associated with longer PFS and trend toward higher response rate (PFS HR, 0.53; 95% CI, 0.28 to 1.00; = .049; OR, 5.88; 95% CI, 0.71 to 4.55; = .09; response rate 33% in TA-high and 7.7% in TA-low).

Conclusion: TA-ness classification is associated with prognosis in patients with mCRC treated with anti-EGFR therapy and may further help understanding the value of sidedness in patients with wild-type tumors.
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http://dx.doi.org/10.1200/PO.20.00050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529528PMC
September 2020

Pertuzumab and trastuzumab emtansine in patients with HER2-amplified metastatic colorectal cancer: the phase II HERACLES-B trial.

ESMO Open 2020 09;5(5):e000911

Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milano, Italy; Dipartimento di Oncologia ed Emato-Oncologia, Università degli Studi di Milano (La Statale), Milano, Italy. Electronic address:

Background: HER2 is a therapeutic target for metastatic colorectal cancer (mCRC), as demonstrated in the pivotal HERACLES-A (HER2 Amplification for Colo-rectaL cancer Enhanced Stratification) trial with trastuzumab and lapatinib. The aim of HERACLES-B trial is to assess the efficacy of the combination of pertuzumab and trastuzumab-emtansine (T-DM1) in this setting.

Methods: HERACLES-B was a single-arm, phase II trial, in patients with histologically confirmed wild-type and HER2+ mCRC refractory to standard treatments. HER2 positivity was assessed by immunohistochemistry and in situ hybridisation according to HERACLES criteria. Patients were treated with pertuzumab (840 mg intravenous load followed by 420 mg intravenous every 3 weeks) and T-DM1 (3.6 mg/kg every 3 weeks) until disease progression or toxicity. Primary and secondary end points were objective response rate (ORR) and progression-free survival (PFS). With a Fleming/Hern design (H0=ORR 10%; α=0.05; power=0.85), 7/30 responses were required to demonstrate an ORR ≥30% (H1).

Results: Thirty-one patients, 48% with ≥4 lines of previous therapies, were treated and evaluable. ORR was 9.7% (95% CI: 0 to 28) and stable disease (SD) 67.7% (95% CI: 50 to 85). OR/SD ≥4 months was associated with higher HER2 immunohistochemistry score (3+ vs 2+) (p = 0.03). Median PFS was 4.1 months (95% CI: 3.6 to 5.9). Drug-related grade (G) 3 adverse events were observed in two patients (thrombocytopaenia); G≤2 AE in 84% of cycles (n = 296), mainly nausea and fatigue.

Conclusions: HERACLES-B trial did not reach its primary end point of ORR; however, based on high disease control, PFS similar to other anti-HER2 regimens, and low toxicity, pertuzumab in combination with T-DM1 can be considered for HER2+mCRC as a potential therapeutic resource.

Trial Registration Number: 2012-002128-33 and NCT03225937.
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http://dx.doi.org/10.1136/esmoopen-2020-000911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523198PMC
September 2020

Long-term Clinical Outcome of Trastuzumab and Lapatinib for HER2-positive Metastatic Colorectal Cancer.

Clin Colorectal Cancer 2020 12 27;19(4):256-262.e2. Epub 2020 Jun 27.

Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy; Dipartimento di Oncologia ed Emato-Oncologia, Università degli Studi di Milano, Milan, Italy. Electronic address:

Background: ERBB2 amplification occurs in 5% of RAS wild-type metastatic colorectal cancer (mCRC) and it has been shown to be a target for treatment with 2 HER2-directed combinations of trastuzumab and lapatinib or trastuzumab and pertuzumab. We present long-term clinical results of trastuzumab and lapatinib (HERACLES-A trial) at 6.7 years (82 months) follow-up and focus on central nervous system (CNS) recurrences.

Patients And Methods: Patients had histologically confirmed KRAS exon 2 (codons 12 and 13) wild-type and HER2-positive mCRC. HER2 positivity was assessed by immunohistochemistry and in situ hybridization HERACLES diagnostic criteria. Patients were treated with intravenous trastuzumab 4 mg/kg loading dose, then 2 mg/kg once per week, and oral lapatinib 1000 mg per day until disease progression or toxicity. Patients who presented with symptoms or signs of CNS disease received brain computed tomography scan or magnetic resonance imaging.

Results: A total of 35 patients received trastuzumab and lapatinib and 32 were evaluable for response. One patient (3%) achieved complete response (CR), 8 (25%) partial response, and 13 (41%) stable disease. Therefore, response rate was 28%. Median progression-free survival was 4.7 months (95% confidence interval [CI] 3.7-6.1). Median overall survival was 10.0 months (95% CI 7.9-15.8). One patient achieved sustained CR still maintained at 7 years of follow-up. Progression in the central nervous system (CNS) occurred in 6 (19%) of 32 patients.

Conclusions: Long-term (6.7 years) follow-up analysis of HERACLES-A supports using of trastuzumab and lapatinib as treatment reference for KRAS wild-type, chemorefractory HER2-positive mCRC. In this subset of patients, prolongation of survival is accompanied by CNS recurrences that will require diagnostic and therapeutic attention in future studies. Clinicaltrials. Gov identifier: NCT 03225937.
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http://dx.doi.org/10.1016/j.clcc.2020.06.009DOI Listing
December 2020

Colorectal cancer residual disease at maximal response to EGFR blockade displays a druggable Paneth cell-like phenotype.

Sci Transl Med 2020 08;12(555)

Department of Oncology, University of Torino, 10060 Candiolo, Torino, Italy.

Blockade of epidermal growth factor receptor (EGFR) causes tumor regression in some patients with metastatic colorectal cancer (mCRC). However, residual disease reservoirs typically remain even after maximal response to therapy, leading to relapse. Using patient-derived xenografts (PDXs), we observed that mCRC cells surviving EGFR inhibition exhibited gene expression patterns similar to those of a quiescent subpopulation of normal intestinal secretory precursors with Paneth cell characteristics. Compared with untreated tumors, these pseudodifferentiated tumor remnants had reduced expression of genes encoding EGFR-activating ligands, enhanced activity of human epidermal growth factor receptor 2 (HER2) and HER3, and persistent signaling along the phosphatidylinositol 3-kinase (PI3K) pathway. Clinically, properties of residual disease cells from the PDX models were detected in lingering tumors of responsive patients and in tumors of individuals who had experienced early recurrence. Mechanistically, residual tumor reprogramming after EGFR neutralization was mediated by inactivation of Yes-associated protein (YAP), a master regulator of intestinal epithelium recovery from injury. In preclinical trials, Pan-HER antibodies minimized residual disease, blunted PI3K signaling, and induced long-term tumor control after treatment discontinuation. We found that tolerance to EGFR inhibition is characterized by inactivation of an intrinsic lineage program that drives both regenerative signaling during intestinal repair and EGFR-dependent tumorigenesis. Thus, our results shed light on CRC lineage plasticity as an adaptive escape mechanism from EGFR-targeted therapy and suggest opportunities to preemptively target residual disease.
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http://dx.doi.org/10.1126/scitranslmed.aax8313DOI Listing
August 2020

Systems analysis of protein signatures predicting cetuximab responses in KRAS, NRAS, BRAF and PIK3CA wild-type patient-derived xenograft models of metastatic colorectal cancer.

Int J Cancer 2020 11 13;147(10):2891-2901. Epub 2020 Aug 13.

Department of Physiology and Medical Physics and Centre Systems Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland.

Antibodies targeting the human epidermal growth factor receptor (EGFR) are used for the treatment of RAS wild-type metastatic colorectal cancer. A significant proportion of patients remains unresponsive to this therapy. Here, we performed a reverse-phase protein array-based (phospho)protein analysis of 63 KRAS, NRAS, BRAF and PIK3CA wild-type metastatic CRC tumours. Responses of tumours to anti-EGFR therapy with cetuximab were recorded in patient-derived xenograft (PDX) models. Unsupervised hierarchical clustering of pretreatment tumour tissue identified three clusters, of which Cluster C3 was exclusively composed of responders. Clusters C1 and C2 exhibited mixed responses. None of the three protein clusters exhibited a significant correlation with transcriptome-based subtypes. Analysis of protein signatures across all PDXs identified 14 markers that discriminated cetuximab-sensitive and cetuximab-resistant tumours: PDK1 (S241), caspase-8, Shc (Y317), Stat3 (Y705), p27, GSK-3β (S9), HER3, PKC-α (S657), EGFR (Y1068), Akt (S473), S6 ribosomal protein (S240/244), HER3 (Y1289), NF-κB-p65 (S536) and Gab-1 (Y627). Least absolute shrinkage and selection operator and binominal logistic regression analysis delivered refined protein signatures for predicting response to cetuximab. (Phospo-)protein analysis of matched pretreated and posttreated models furthermore showed significant reduction of Gab-1 (Y627) and GSK-3β (S9) exclusively in responding models, suggesting novel targets for treatment.
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http://dx.doi.org/10.1002/ijc.33226DOI Listing
November 2020

Innovations, challenges, and minimal information for standardization of humanized mice.

EMBO Mol Med 2020 07 24;12(7):e8662. Epub 2020 Jun 24.

The Jackson Laboratory, Bar Harbor, ME, USA.

Mice xenotransplanted with human cells and/or expressing human gene products (also known as "humanized mice") recapitulate the human evolutionary specialization and diversity of genotypic and phenotypic traits. These models can provide a relevant in vivo context for understanding of human-specific physiology and pathologies. Humanized mice have advanced toward mainstream preclinical models and are now at the forefront of biomedical research. Here, we considered innovations and challenges regarding the reconstitution of human immunity and human tissues, modeling of human infections and cancer, and the use of humanized mice for testing drugs or regenerative therapy products. As the number of publications exploring different facets of humanized mouse models has steadily increased in past years, it is becoming evident that standardized reporting is needed in the field. Therefore, an international community-driven resource called "Minimal Information for Standardization of Humanized Mice" (MISHUM) has been created for the purpose of enhancing rigor and reproducibility of studies in the field. Within MISHUM, we propose comprehensive guidelines for reporting critical information generated using humanized mice.
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http://dx.doi.org/10.15252/emmm.201708662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338801PMC
July 2020

EGFR Blockade Reverts Resistance to KRAS Inhibition in Colorectal Cancer.

Cancer Discov 2020 08 19;10(8):1129-1139. Epub 2020 May 19.

Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, New York.

Most patients with -mutant non-small cell lung cancer (NSCLC) experience clinical benefit from selective KRAS inhibition, whereas patients with colorectal cancer bearing the same mutation rarely respond. To investigate the cause of the limited efficacy of KRAS inhibitors in colorectal cancer, we examined the effects of AMG510 in colorectal cancer cell lines. Unlike NSCLC cell lines, colorectal cancer models have high basal receptor tyrosine kinase (RTK) activation and are responsive to growth factor stimulation. In colorectal cancer lines, KRAS inhibition induces higher phospho-ERK rebound than in NSCLC cells. Although upstream activation of several RTKs interferes with KRAS blockade, we identify EGFR signaling as the dominant mechanism of colorectal cancer resistance to KRAS inhibitors. The combinatorial targeting of EGFR and KRAS is highly effective in colorectal cancer cells and patient-derived organoids and xenografts, suggesting a novel therapeutic strategy to treat patients with colorectal cancer. SIGNIFICANCE: The efficacy of KRAS inhibitors in NSCLC and colorectal cancer is lineage-specific. RTK dependency and signaling rebound kinetics are responsible for sensitivity or resistance to KRAS inhibition in colorectal cancer. EGFR and KRAS should be concomitantly inhibited to overcome resistance to KRAS blockade in colorectal tumors...
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http://dx.doi.org/10.1158/2159-8290.CD-20-0187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416460PMC
August 2020

Vitamin C Restricts the Emergence of Acquired Resistance to EGFR-Targeted Therapies in Colorectal Cancer.

Cancers (Basel) 2020 Mar 14;12(3). Epub 2020 Mar 14.

Candiolo Cancer Institute, FPO-IRCCS, Candiolo 10060 (TO), Italy.

The long-term efficacy of the Epidermal Growth Factor Receptor (EGFR)-targeted antibody cetuximab in advanced colorectal cancer (CRC) patients is limited by the emergence of drug-resistant (persister) cells. Recent studies in other cancer types have shown that cells surviving initial treatment with targeted agents are often vulnerable to alterations in cell metabolism including oxidative stress. Vitamin C (VitC) is an antioxidant agent which can paradoxically trigger oxidative stress at pharmacological dose. Here we tested the hypothesis that VitC in combination with cetuximab could restrain the emergence of secondary resistance to EGFR blockade in CRC wild-type models. We found that addition of VitC to cetuximab impairs the emergence of drug persisters, limits the growth of CRC organoids, and significantly delays acquired resistance in CRC patient-derived xenografts. Mechanistically, proteomic and metabolic flux analysis shows that cetuximab blunts carbohydrate metabolism by blocking glucose uptake and glycolysis, beyond promoting slow but progressive ROS production. In parallel, VitC disrupts iron homeostasis and further increases ROS levels ultimately leading to ferroptosis. Combination of VitC and cetuximab orchestrates a synthetic lethal metabolic cell death program triggered by ATP depletion and oxidative stress, which effectively limits the emergence of acquired resistance to anti-EGFR antibodies. Considering that high-dose VitC is known to be safe in cancer patients, our findings might have clinical impact on CRC patients treated with anti-EGFR therapies.
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http://dx.doi.org/10.3390/cancers12030685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140052PMC
March 2020

Immunogenomics of Colorectal Tumors: Facts and Hypotheses on an Evolving Saga.

Trends Cancer 2019 12 6;5(12):779-788. Epub 2019 Nov 6.

Department of Oncology, University of Torino, 10060 Candiolo, Torino, Italy; Candiolo Cancer Institute - FPO IRCCS, 10060 Candiolo, Torino, Italy. Electronic address:

Immunotherapy with immune checkpoint inhibitors is an approved treatment option for a subpopulation of patients with colorectal cancers that display microsatellite instability. However, not all individuals within this subgroup respond to immunotherapy, and molecular biomarkers for effective patient stratification are still lacking. In this opinion article, we provide an overview of the different biological parameters that contribute to rendering colorectal cancers with microsatellite instability potentially sensitive to immunotherapy. We critically discuss the reasons why such parameters have limited predictive value and the implications therein. We also consider that a more informed knowledge of response determinants in this tumor subtype could help understand the mechanisms of immunotherapy resistance in microsatellite stable tumors. We conclude that the dynamic nature of the interactions between cancer and immune cells complicates conventional biomarker development and argue that a new generation of adaptive metrics, borrowed from evolutionary genetics, may improve the effectiveness and reliability of clinical decision making.
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http://dx.doi.org/10.1016/j.trecan.2019.10.006DOI Listing
December 2019

Adaptive mutability of colorectal cancers in response to targeted therapies.

Science 2019 12 7;366(6472):1473-1480. Epub 2019 Nov 7.

Candiolo Cancer Institute, FPO-IRCCS, Candiolo (TO) 10060, Italy.

The emergence of drug resistance limits the efficacy of targeted therapies in human tumors. The prevalent view is that resistance is a fait accompli: when treatment is initiated, cancers already contain drug-resistant mutant cells. Bacteria exposed to antibiotics transiently increase their mutation rates (adaptive mutability), thus improving the likelihood of survival. We investigated whether human colorectal cancer (CRC) cells likewise exploit adaptive mutability to evade therapeutic pressure. We found that epidermal growth factor receptor (EGFR)/BRAF inhibition down-regulates mismatch repair (MMR) and homologous recombination DNA-repair genes and concomitantly up-regulates error-prone polymerases in drug-tolerant (persister) cells. MMR proteins were also down-regulated in patient-derived xenografts and tumor specimens during therapy. EGFR/BRAF inhibition induced DNA damage, increased mutability, and triggered microsatellite instability. Thus, like unicellular organisms, tumor cells evade therapeutic pressures by enhancing mutability.
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http://dx.doi.org/10.1126/science.aav4474DOI Listing
December 2019

Patient-Derived Xenografts and Matched Cell Lines Identify Pharmacogenomic Vulnerabilities in Colorectal Cancer.

Clin Cancer Res 2019 10 2;25(20):6243-6259. Epub 2019 Aug 2.

Department of Oncology, University of Torino, Candiolo, Torino, Italy.

Purpose: Patient-derived xenograft (PDX) models accurately recapitulate the tumor of origin in terms of histopathology, genomic landscape, and therapeutic response, but some limitations due to costs associated with their maintenance and restricted amenability for large-scale screenings still exist. To overcome these issues, we established a platform of 2D cell lines (xeno-cell lines, XL), derived from PDXs of colorectal cancer with matched patient germline gDNA available.

Experimental Design: Whole-exome and transcriptome sequencing analyses were performed. Biomarkers of response and resistance to anti-HER therapy were annotated. Dependency on the helicase gene was assessed in MSS, MSI-H, and MSI-like XLs using a reverse genetics functional approach.

Results: XLs recapitulated the entire spectrum of colorectal cancer transcriptional subtypes. Exome and RNA-seq analyses delineated several molecular biomarkers of response and resistance to EGFR and HER2 blockade. Genotype-driven responses observed in XLs were confirmed in the matched PDXs. MSI-H models were dependent upon gene expression, while loss of did not affect MSS XLs growth. Interestingly, one MSS XL with transcriptional MSI-like traits was sensitive to depletion.

Conclusions: The XL platform represents a preclinical tool for functional gene validation and proof-of-concept studies to identify novel druggable vulnerabilities in colorectal cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611232PMC
October 2019

The Stromal and Immune Landscape of Colorectal Cancer Progression during Anti-EGFR Therapy.

Cancer Cell 2019 07;36(1):1-3

Department of Oncology, University of Torino, 10060 Candiolo, Torino, Italy; Candiolo Cancer Institute-FPO IRCCS, 10060 Candiolo, Torino, Italy. Electronic address:

In this issue of Cancer Cell, Woolston et al. show that colorectal cancers that become refractory to initially effective anti-EGFR therapy contain an abundance of stromal and immune cells, irrespective of the contextual presence of resistance-conferring mutations. This reconfiguration puts forward therapeutic opportunities for patients who relapse on EGFR-targeting treatment.
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http://dx.doi.org/10.1016/j.ccell.2019.06.001DOI Listing
July 2019

Evolving neoantigen profiles in colorectal cancers with DNA repair defects.

Genome Med 2019 06 28;11(1):42. Epub 2019 Jun 28.

Candiolo Cancer Institute, FPO-IRCCS, 10060, Candiolo (TO), Italy.

Background: Neoantigens that arise as a consequence of tumor-specific mutations can be recognized by T lymphocytes leading to effective immune surveillance. In colorectal cancer (CRC) and other tumor types, a high number of neoantigens is associated with patient response to immune therapies. The molecular processes governing the generation of neoantigens and their turnover in cancer cells are poorly understood. We exploited CRC as a model system to understand how alterations in DNA repair pathways modulate neoantigen profiles over time.

Methods: We performed whole exome sequencing (WES) and RNA sequencing (RNAseq) in CRC cell lines, in vitro and in vivo, and in CRC patient-derived xenografts (PDXs) to track longitudinally genomic profiles, clonal evolution, mutational signatures, and predicted neoantigens.

Results: The majority of CRC models showed remarkably stable mutational and neoantigen profiles; however, those carrying defects in DNA repair genes continuously diversified. Rapidly evolving and evolutionary stable CRCs displayed characteristic genomic signatures and transcriptional profiles. Downregulation of molecules implicated in antigen presentation occurred selectively in highly mutated and rapidly evolving CRC.

Conclusions: These results indicate that CRCs carrying alterations in DNA repair pathways display dynamic neoantigen patterns that fluctuate over time. We define CRC subsets characterized by slow and fast evolvability and link this phenotype to downregulation of antigen-presenting cellular mechanisms. Longitudinal monitoring of the neoantigen landscape could be relevant in the context of precision medicine.
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http://dx.doi.org/10.1186/s13073-019-0654-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599263PMC
June 2019

Combined blockade of MEK and PI3KCA as an effective antitumor strategy in HER2 gene amplified human colorectal cancer models.

J Exp Clin Cancer Res 2019 Jun 4;38(1):236. Epub 2019 Jun 4.

Medical Oncology, Department of Precision Medicine, Università degli Studi della Campania "Luigi Vanvitelli", Via S. Pansini 5, 80131, Naples, Italy.

Background: Targeting the epidermal growth factor receptor (EGFR) either alone or in combination with chemotherapy is an effective treatment for patients with RAS wild-type metastatic colorectal cancer (mCRC). However, only a small percentage of mCRC patients receive clinical benefits from anti-EGFR therapies, due to the development of resistance mechanisms. In this regard, HER2 has emerged as an actionable target in the treatment of mCRC patients with resistance to anti-EGFR therapy.

Methods: We have used SW48 and LIM1215 human colon cancer cell lines, quadruple wild-type for KRAS, NRAS, BRAF and PI3KCA genes, and their HER2-amplified (LIM1215-HER2 and SW48-HER2) derived cells to perform in vitro and in vivo studies in order to identify novel therapeutic strategies in HER2 gene amplified human colorectal cancer.

Results: LIM1215-HER2 and SW48-HER2 cells showed over-expression and activation of the HER family receptors and concomitant intracellular downstream signaling including the pro-survival PI3KCA/AKT and the mitogenic RAS/RAF/MEK/MAPK pathways. HER2-amplified cells were treated with several agents including anti-EGFR antibodies (cetuximab, SYM004 and MM151); anti-HER2 (trastuzumab, pertuzumab and lapatinib) inhibitors; anti-HER3 (duligotuzumab) inhibitors; and MEK and PI3KCA inhibitors, such as refametinib and pictilisib, as single agents and in combination. Subsequently, different in vivo experiments have been performed. MEK plus PI3KCA inhibitors treatment determined the best antitumor activity. These results were validated in vivo in HER2-amplified patient derived tumor xenografts from three metastatic colorectal cancer patients.

Conclusions: These results suggest that combined therapy with MEK and PI3KCA inhibitors could represent a novel and effective treatment option for HER2-amplified colorectal cancer.
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http://dx.doi.org/10.1186/s13046-019-1230-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6549349PMC
June 2019

INKA, an integrative data analysis pipeline for phosphoproteomic inference of active kinases.

Mol Syst Biol 2019 04 12;15(4):e8250. Epub 2019 Apr 12.

Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands

Identifying hyperactive kinases in cancer is crucial for individualized treatment with specific inhibitors. Kinase activity can be discerned from global protein phosphorylation profiles obtained with mass spectrometry-based phosphoproteomics. A major challenge is to relate such profiles to specific hyperactive kinases fueling growth/progression of tumors. Hitherto, the focus has been on phosphorylation of either kinases or their substrates. Here, we combined label-free kinase-centric and substrate-centric information in an Integrative Inferred Kinase Activity (INKA) analysis. This multipronged, stringent analysis enables ranking of kinase activity and visualization of kinase-substrate networks in a single biological sample. To demonstrate utility, we analyzed (i) cancer cell lines with known oncogenes, (ii) cell lines in a differential setting (wild-type versus mutant, +/- drug), (iii) pre- and on-treatment tumor needle biopsies, (iv) cancer cell panel with available drug sensitivity data, and (v) patient-derived tumor xenografts with INKA-guided drug selection and testing. These analyses show superior performance of INKA over its components and substrate-based single-sample tool KARP, and underscore target potential of high-ranking kinases, encouraging further exploration of INKA's functional and clinical value.
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http://dx.doi.org/10.15252/msb.20188250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461034PMC
April 2019
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