Tumour mutational burden and survival with molecularly matched therapy.

BACKGROUND: The impact of tumour mutational burden (TMB) on outcome with molecularly matched therapy is unknown. Higher TMB could predict resistance to molecularly matched therapy through co-occurring driver mutations. METHODS: One hundred and four patients with advanced cancers underwent molecular profiling in the DKTK-MASTER program. Fifty-five patients received systemic therapy excluding immunotherapy. Patients with molecularly matched (n = 35) or non-molecularly informed therapy (n = 20) were analysed for TMB and survival. Results were validated in an independent cohort of patients receiving molecularly matched (n = 68) or non-molecularly informed therapy (n = 40). Co-occurring driver mutations and TMB were analysed in the exploratory cohort and The Cancer Genome Atlas (TCGA) datasets. RESULTS: Patients were stratified by the median TMB of 1.67 mutations per Megabase (mut/Mb) of 35 patients receiving molecularly matched therapy into TMB-high or TMB-low groups. Median overall survival (4 months [95% CI, 3.3-7.6] versus 12.8 months [95% CI, 10-not reached], p < 0.001) and progression-free survival (1.8 months [95% CI, 1.1-3.7] versus 7.9 months [95% CI, 2.8-17.0], p = 0.003) were significantly shorter in the TMB-high group compared to the TMB-low group. In the validation cohort, shorter OS and PFS were identified in the TMB-high group (TMB cut-off of 4 mut/Mb) treated with molecularly matched therapy. No differences were observed in patients receiving non-molecularly informed systemic therapy. A significant correlation between co-occurring driver mutations and TMB (n = 104, r = 0.78 [95% CI, 0.68-0.85], p < 0.001) was found in the exploratory cohort as well as the majority (24/33) of TCGA studies. CONCLUSION: A high TMB was associated with unfavourable outcome in patients receiving molecularly matched therapy, indicating untargeted resistance pathways. Therefore, TMB should be further investigated as a predictive biomarker in precision oncology programs.

The FGFR inhibitor PD173074 binds to the C-terminus of oncofetal HMGA2 and modulates its DNA-binding and transcriptional activation functions.

The architectural chromatin factor high-mobility group AT-hook 2 (HMGA2) is causally involved in several human malignancies and pathologies. HMGA2 is not expressed in most normal adult somatic cells, which renders the protein an attractive drug target. An established cell-based compound library screen identified the fibroblast growth factor receptor (FGFR) inhibitor PD173074 as an antagonist of HMGA2-mediated transcriptional reporter gene activation. We determined that PD173074 binds the C-terminus of HMGA2 and interferes with functional coordination of the three AT-hook DNA-binding domains mediated by the C-terminus. The HMGA2-antagonistic effect of PD173074 on transcriptional activation may therefore result from an induced altered DNA-binding mode of HMGA2. PD173074 as a novel HMGA2-specific antagonist could trigger the development of derivates with enhanced attributes and clinical potential.

A combined computational and functional approach identifies IGF2BP2 as a driver of chemoresistance in a wide array of pre-clinical models of colorectal cancer.

AIM: Chemoresistance is a major cause of treatment failure in colorectal cancer (CRC) therapy. In this study, the impact of the IGF2BP family of RNA-binding proteins on CRC chemoresistance was investigated using in silico, in vitro, and in vivo approaches. METHODS: Gene expression data from a well-characterized cohort and publicly available cross-linking immunoprecipitation sequencing (CLIP-Seq) data were collected. Resistance to chemotherapeutics was assessed in patient-derived xenografts (PDXs) and patient-derived organoids (PDOs). Functional studies were performed in 2D and 3D cell culture models, including proliferation, spheroid growth, and mitochondrial respiration analyses. RESULTS: We identified IGF2BP2 as the most abundant IGF2BP in primary and metastastatic CRC, correlating with tumor stage in patient samples and tumor growth in PDXs. IGF2BP2 expression in primary tumor tissue was significantly associated with resistance to selumetinib, gefitinib, and regorafenib in PDOs and to 5-fluorouracil and oxaliplatin in PDX in vivo. IGF2BP2 knockout (KO) HCT116 cells were more susceptible to regorafenib in 2D and to oxaliplatin, selumitinib, and nintedanib in 3D cell culture. Further, a bioinformatic analysis using CLIP data suggested stabilization of target transcripts in primary and metastatic tumors. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) revealed a decreased basal OCR and an increase in glycolytic ATP production rate in IGF2BP2 KO. In addition, real-time reverse transcriptase polymerase chain reaction (qPCR) analysis confirmed decreased expression of genes of the respiratory chain complex I, complex IV, and the outer mitochondrial membrane in IGF2BP2 KO cells. CONCLUSIONS: IGF2BP2 correlates with CRC tumor growth in vivo and promotes chemoresistance by altering mitochondrial respiratory chain metabolism. As a druggable target, IGF2BP2 could be used in future CRC therapy to overcome CRC chemoresistance.

Silencing effects of mutant RAS signalling on transcriptomes.

Mutated genes of the RAS family encoding small GTP-binding proteins drive numerous cancers, including pancreatic, colon and lung tumors. Besides the numerous effects of mutant RAS gene expression on aberrant proliferation, transformed phenotypes, metabolism, and therapy resistance, the most striking consequences of chronic RAS activation are changes of the genetic program. By performing systematic gene expression studies in cellular models that allow comparisons of pre-neoplastic with RAS-transformed cells, we and others have estimated that 7 percent or more of all transcripts are altered in conjunction with the expression of the oncogene. In this context, the number of up-regulated transcripts approximates that of down-regulated transcripts. While up-regulated transcription factors such as MYC, FOSL1, and HMGA2 have been identified and characterized as RAS-responsive drivers of the altered transcriptome, the suppressed factors have been less well studied as potential regulators of the genetic program and transformed phenotype in the breadth of their occurrence. We therefore have collected information on downregulated RAS-responsive factors and discuss their potential role as tumor suppressors that are likely to antagonize active cancer drivers. To better understand the active mechanisms that entail anti-RAS function and those that lead to loss of tumor suppressor activity, we focus on the tumor suppressor HREV107 (alias PLAAT3 [Phospholipase A and acyltransferase 3], PLA2G16 [Phospholipase A2, group XVI] and HRASLS3 [HRAS-like suppressor 3]). Inactivating HREV107 mutations in tumors are extremely rare, hence epigenetic causes modulated by the RAS pathway are likely to lead to down-regulation and loss of function.

Serial Analysis of Gene Mutations and Gene Expression during First-Line Chemotherapy against Metastatic Colorectal Cancer: Identification of Potentially Actionable Targets within the Multicenter Prospective Biomarker Study REVEAL.

Most metastatic colorectal cancer (mCRC) patients succumb to refractory disease due to secondary chemotherapy resistance. To elucidate the molecular changes associated with secondary resistance, we recruited 64 patients with mCRC and hepatic metastases before standard first-line chemotherapy between 2014 and 2018. We subjected DNA from primary tumor specimens (P), hepatic metastasis specimens after treatment (M), and liquid biopsies (L) taken prior to (pre), during (intra), and after (post) treatment to next generation sequencing. We performed Nanostring expression analysis in P and M specimens. Comparative bioinformatics and statistical analysis revealed typical mutational patterns with frequent alterations in TP53, APC, and KRAS in P specimens (n = 48). P and pre-L (n = 42), as well as matched P and M (n = 30), displayed a similar mutation spectrum. In contrast, gene expression profiles classified P (n = 31) and M (n = 23), distinguishable by up-regulation of immune/cytokine receptor and autophagy programs. Switching of consensus molecular subtypes from P to M occurred in 58.3% of cases. M signature genes SFRP2 and SPP1 associated with inferior survival, as validated in an independent cohort. Molecular changes during first-line treatment were detectable by expression profiling rather than by mutational tumor and liquid biopsy analyses. SFRP2 and SPP1 may serve as biomarkers and/or actionable targets.

IC50: an unsuitable measure for large-sized prostate cancer spheroids in drug sensitivity evaluation.

Preclinical models of tumors have the potential to become valuable tools for commercial drug research and development, and 3D culture systems are gaining traction in this area, particularly in prostate cancer (PCa) research. However, nearly all 3D drug design and screening assessments are based on 2D experiments, suggesting limitations of 3D drug testing. To simulate the natural response of human cells to the drug, we detected the half-maximal inhibitory concentration (IC50) changes of 2D/3D LNCaP cells in the drug docetaxel, as well as the sensitivity of different morphologies of 2D/3D LNCaP to docetaxel treatment. In contrast to 2D LNCaP cells, the evaluation of LNCaP spheroids' susceptibility to treatment was more complicated; the fitness of IC50 curves of 2D and 3D tumor cell preclinical models differs significantly. IC50 curves were unsuitable for large-sized LNCaP spheroids. More evaluation indexes (such as max inhibition) and experiments (such as spheroids formation) should be explored and performed to evaluate the susceptibility systematically.

Identification of a neural development gene expression signature in colon cancer stem cells reveals a role for EGR2 in tumorigenesis.

Recent evidence demonstrates that colon cancer stem cells (CSCs) can generate neurons that synapse with tumor innervating fibers required for tumorigenesis and disease progression. Greater understanding of the mechanisms that regulate CSC driven tumor neurogenesis may therefore lead to more effective treatments. RNA-sequencing analyses of ALDHPositive CSCs from colon cancer patient-derived organoids (PDOs) and xenografts (PDXs) showed CSCs to be enriched for neural development genes. Functional analyses of genes differentially expressed in CSCs from PDO and PDX models demonstrated the neural crest stem cell (NCSC) regulator EGR2 to be required for tumor growth and to control expression of homebox superfamily embryonic master transcriptional regulator HOX genes and the neural stem cell and master cell fate regulator SOX2. These data support CSCs as the source of tumor neurogenesis and suggest that targeting EGR2 may provide a therapeutic differentiation strategy to eliminate CSCs and block nervous system driven disease progression.

Biomarker-driven therapies for metastatic uveal melanoma: A prospective precision oncology feasibility study.

BACKGROUND: Targeted therapies for metastatic uveal melanoma have shown limited benefit in biomarker-unselected populations. The Treat20 Plus study prospectively evaluated the feasibility of a precision oncology strategy in routine clinical practice. PATIENTS AND METHODS: Fresh biopsies were analyzed by high-throughput genomics (whole-genome, whole-exome, and RNA sequencing). A multidisciplinary molecular and immunologic tumor board (MiTB) made individualized treatment recommendations based on identified molecular aberrations, patient situation, drug, and clinical trial availability. Therapy selection was at the discretion of the treating physician. The primary endpoint was the feasibility of the precision oncology clinical program. RESULTS: Molecular analyses were available for 39/45 patients (87%). The MiTB provided treatment recommendations for 40/45 patients (89%), of whom 27/45 (60%) received ≥1 matched therapy. First-line matched therapies included MEK inhibitors (n = 15), MET inhibitors (n = 10), sorafenib (n = 1), and nivolumab (n = 1). The best response to first-line matched therapy was partial response in one patient (nivolumab based on tumor mutational burden), mixed response in two patients, and stable disease in 12 patients for a clinical benefit of 56%. The matched therapy population had a median progression-free survival and overall survival of 3.3 and 13.9 months, respectively. The growth modulation index with matched therapy was >1.33 in 6/17 patients (35%) with prior systemic therapy, suggesting clinical benefit. CONCLUSIONS: A precision oncology approach was feasible for patients with metastatic uveal melanoma, with 60% receiving a therapy matched to identify molecular aberrations. The clinical benefit after checkpoint inhibitors highlights the value of tumor mutational burden testing.

Evaluation of JQ1 Combined With Docetaxel for the Treatment of Prostate Cancer Cells in 2D- and 3D-Culture Systems.

Introduction: Prostate cancer (PCa) is dependent on coupled androgen-androgen receptor (AR) signaling for growth and progression. Significant efforts have been made in this research field, as hormonal therapies have greatly improved the survival of patients with metastatic PCa (mPCa). The drug treatment agent JQ1, which potently abrogates bromodomain 4 (BRD4) localization to the AR target loci and therefore significantly impairs AR-mediated gene transcription, is a potent therapeutic option for patients with advanced PCa. In this study, we aimed to investigate the inhibitory effect of JQ1 combined with docetaxel on PCa cells in vitro for the first time. Furthermore, the 3D spheroid culture system was modeled to more accurately simulate the response of PCa cells to drugs. Methods: We established and measured 3D LNCaP spheroids in vitro in order to evaluate the susceptibility of 2D- and 3D-cultured LNCaP cells exposed to the same anti-cancer drug. Results: We demonstrated that JQ1 was an effective drug for promoting cell inhibition after docetaxel treatment in 2D- and 3D- cultured LNCaP cells. Inhibition of 3D cultured formation in the combined treatment group was significantly higher than that in docetaxel or JQ1 alone. Under the same conditions of drug solubility, the drug resistance of 3D spheroids was significantly higher than that of 2D cells. Moreover, dmax and lg volume were suitable parameters for LNCaP cells/spheroid size displaying and evaluating cell viability. Conclusion: 3D cultured spheroids of PCa are an effective tool for studying PCa drug trials. JQ1 combined with docetaxel may be an effective treatment for advanced PCa. This combination therapy strategy deserves further evaluation in clinical trials.

Rapid testing of candidate oncogenes and tumour suppressor genes in signal transduction and neoplastic transformation.

The COSMIC database (version 94) lists 576 genes in the Cancer Gene Census which have a defined function as drivers of malignancy (oncogenes) or as tumour suppressors (Tier 1). In addition, there are 147 genes with similar functions, but which are less well characterised (Tier 2). Furthermore, next-generation sequencing projects in the context of precision oncology activities are constantly discovering new ones. Since cancer genes differ from their wild-type precursors in numerous molecular and biochemical properties and exert significant differential effects on downstream processes, simple assays that can uncover oncogenic or anti-oncogenic functionality are desirable and may precede more sophisticated analyses. We describe simple functional assays for PTPN11 (protein-tyrosine phosphatase, non-receptor-type 11)/SHP2 mutants, which are typically found in RASopathies and exhibit potential oncogenic activity. We have also designed a functional test for lysyl oxidase (LOX), a prototypical class II tumour suppressor gene whose loss of function may contribute to neoplastic transformation by RAS oncogenes. Moreover, we applied this test to analyse three co-regulated, RAS-responsive genes for transformation-suppressive activity. The integration of these tests into systems biology studies will contribute to a better understanding of cellular networks in cancer.

RNA sequencing of long-term label-retaining colon cancer stem cells identifies novel regulators of quiescence.

Recent data suggest that therapy-resistant quiescent cancer stem cells (qCSCs) are the source of relapse in colon cancer. Here, using colon cancer patient-derived organoids and xenografts, we identify rare long-term label-retaining qCSCs that can re-enter the cell cycle to generate new tumors. RNA sequencing analyses demonstrated that these cells display the molecular hallmarks of quiescent tissue stem cells, including expression of p53 signaling genes, and are enriched for transcripts common to damage-induced quiescent revival stem cells of the regenerating intestine. In addition, we identify negative regulators of cell cycle, downstream of p53, that we show are indicators of poor prognosis and may be targeted for qCSC abolition in both p53 wild-type and mutant tumors. These data support the temporal inhibition of downstream targets of p53 signaling, in combination with standard-of-care treatments, for the elimination of qCSCs and prevention of relapse in colon cancer.

Generation of Patient-Derived Colorectal Cancer Organoids for RAS Studies.

Human cell line models have been widely used for testing of novel anticancer compounds and for predicting clinical response to monotherapies and combinatorial therapies. For many years, standard monolayer culture conditions were used as gold standard, only surpassed by in vivo testing of mouse models. Recently, the incorporation of three-dimensional culture has been shown to further improve predictive compound testing. In view of the renewed interest in anti-RAS cancer therapy, we provide a protocol for establishing colorectal cancer organoids which are characterized by a high prevalence of KRAS mutations.

Mutation-specific effects of NRAS oncogenes in colorectal cancer cells.

In colorectal cancer (CRC), the prevalence of NRAS mutations (5-9%) is inferior to that of KRAS mutations (40-50%). NRAS mutations feature lately during tumour progression and drive resistance to anti-EGFR therapy in KRAS wild-type tumours. To elucidate specific functions of NRAS mutations in CRC, we expressed doxycycline-inducible G12D and Q61K mutations in the CRC cell line Caco-2. A focused phospho-proteome analysis based on the Bio-Plex platform, which interrogated the activity of MAPK, PI3K, mTOR, STAT, p38, JNK and ATF2, did not reveal significant differences between Caco-2 cells expressing NRASG12D, NRASQ61K and KRASG12V. However, phenotypic read-outs were different. The NRAS Q61K mutation promoted anchorage-independent proliferation and tumorigenicity, similar to features driven by canonical KRAS mutations. In contrast, expression of NRASG12D resulted in reduced proliferation and apoptosis. At the transcriptome level, we saw upregulation of cytokines and chemokines. IL1A, IL11, CXCL8 (IL-8) and CCL20 exhibited enhanced secretion into the culture medium. In addition, RNA sequencing results indicated activation of the IL1-, JAK/STAT-, NFκB- and TNFα signalling pathways. These results form the basis for an NRASG12D-driven inflammatory phenotype in CRC.

SFPQ Depletion Is Synthetically Lethal with BRAFV600E in Colorectal Cancer Cells.

Oncoproteins such as the BRAFV600E kinase endow cancer cells with malignant properties, but they also create unique vulnerabilities. Targeting of BRAFV600E-driven cytoplasmic signaling networks has proved ineffective, as patients regularly relapse with reactivation of the targeted pathways. We identify the nuclear protein SFPQ to be synthetically lethal with BRAFV600E in a loss-of-function shRNA screen. SFPQ depletion decreases proliferation and specifically induces S-phase arrest and apoptosis in BRAFV600E-driven colorectal and melanoma cells. Mechanistically, SFPQ loss in BRAF-mutant cancer cells triggers the Chk1-dependent replication checkpoint, results in decreased numbers and reduced activities of replication factories, and increases collision between replication and transcription. We find that BRAFV600E-mutant cancer cells and organoids are sensitive to combinations of Chk1 inhibitors and chemically induced replication stress, pointing toward future therapeutic approaches exploiting nuclear vulnerabilities induced by BRAFV600E.

Reduced replication origin licensing selectively kills KRAS-mutant colorectal cancer cells via mitotic catastrophe.

To unravel vulnerabilities of KRAS-mutant CRC cells, a shRNA-based screen specifically inhibiting MAPK pathway components and targets was performed in CaCo2 cells harboring conditional oncogenic KRASG12V. The custom-designed shRNA library comprised 121 selected genes, which were previously identified to be strongly regulated in response to MEK inhibition. The screen showed that CaCo2 cells expressing KRASG12V were sensitive to the suppression of the DNA replication licensing factor minichromosome maintenance complex component 7 (MCM7), whereas KRASwt CaCo2 cells were largely resistant to MCM7 suppression. Similar results were obtained in an isogenic DLD-1 cell culture model. Knockdown of MCM7 in a KRAS-mutant background led to replication stress as indicated by increased nuclear RPA focalization. Further investigation showed a significant increase in mitotic cells after simultaneous MCM7 knockdown and KRASG12V expression. The increased percentage of mitotic cells coincided with strongly increased DNA damage in mitosis. Taken together, the accumulation of DNA damage in mitotic cells is due to replication stress that remained unresolved, which results in mitotic catastrophe and cell death. In summary, the data show a vulnerability of KRAS-mutant cells towards suppression of MCM7 and suggest that inhibiting DNA replication licensing might be a viable strategy to target KRAS-mutant cancers.

Support of a molecular tumour board by an evidence-based decision management system for precision oncology.

BACKGROUND: Reliable and reproducible interpretation of molecular aberrations constitutes a bottleneck of precision medicine. Evidence-based decision management systems may improve rational therapy recommendations. To cope with an increasing amount of complex molecular data in the clinical care of patients with cancer, we established a workflow for the interpretation of molecular analyses. METHODS: A specialized physician screened results from molecular analyses for potential biomarkers, irrespective of the diagnostic modality. Best available evidence was retrieved and categorized through establishment of an in-house database and interrogation of publicly available databases. Annotated biomarkers were ranked using predefined evidence levels and subsequently discussed at a molecular tumour board (MTB), which generated treatment recommendations. Subsequent translation into patient treatment and clinical outcomes were followed up. RESULTS: One hundred patients were discussed in the MTB between January 2016 and May 2017. Molecular data were obtained for 70 of 100 patients (50 whole exome/RNA sequencing, 18 panel sequencing, 2 immunohistochemistry (IHC)/microsatellite instability analysis). The MTB generated a median of two treatment recommendations each for 63 patients. Thirty-nine patients were treated: 6 partial responses and 12 stable diseases were achieved as best responses. Genetic counselling for germline events was recommended for seven patients. CONCLUSION: The development of an evidence-based workflow allowed for the clinical interpretation of complex molecular data and facilitated the translation of personalized treatment strategies into routine clinical care. The high number of treatment recommendations in patients with comprehensive genomic data and promising responses in patients treated with combination therapy warrant larger clinical studies.

VIST - a Variant-Information Search Tool for precision oncology.

BACKGROUND: Diagnosis and treatment decisions in cancer increasingly depend on a detailed analysis of the mutational status of a patient's genome. This analysis relies on previously published information regarding the association of variations to disease progression and possible interventions. Clinicians to a large degree use biomedical search engines to obtain such information; however, the vast majority of scientific publications focus on basic science and have no direct clinical impact. We develop the Variant-Information Search Tool (VIST), a search engine designed for the targeted search of clinically relevant publications given an oncological mutation profile. RESULTS: VIST indexes all PubMed abstracts and content from ClinicalTrials.gov. It applies advanced text mining to identify mentions of genes, variants and drugs and uses machine learning based scoring to judge the clinical relevance of indexed abstracts. Its functionality is available through a fast and intuitive web interface. We perform several evaluations, showing that VIST's ranking is superior to that of PubMed or a pure vector space model with regard to the clinical relevance of a document's content. CONCLUSION: Different user groups search repositories of scientific publications with different intentions. This diversity is not adequately reflected in the standard search engines, often leading to poor performance in specialized settings. We develop a search engine for the specific case of finding documents that are clinically relevant in the course of cancer treatment. We believe that the architecture of our engine, heavily relying on machine learning algorithms, can also act as a blueprint for search engines in other, equally specific domains. VIST is freely available at https://vist.informatik.hu-berlin.de/.

Discovery and Validation of Novel Biomarkers for Detection of Epithelial Ovarian Cancer.

Detection of epithelial ovarian cancer (EOC) poses a critical medical challenge. However, novel biomarkers for diagnosis remain to be discovered. Therefore, innovative approaches are of the utmost importance for patient outcome. Here, we present a concept for blood-based biomarker discovery, investigating both epithelial and specifically stromal compartments, which have been neglected in search for novel candidates. We queried gene expression profiles of EOC including microdissected epithelium and adjacent stroma from benign and malignant tumours. Genes significantly differentially expressed within either the epithelial or the stromal compartments were retrieved. The expression of genes whose products are secreted yet absent in the blood of healthy donors were validated in tissue and blood from patients with pelvic mass by NanoString analysis. Results were confirmed by the comprehensive gene expression database, CSIOVDB (Ovarian cancer database of Cancer Science Institute Singapore). The top 25% of candidate genes were explored for their biomarker potential, and twelve were able to discriminate between benign and malignant tumours on transcript levels (p < 0.05). Among them T-cell differentiation protein myelin and lymphocyte (MAL), aurora kinase A (AURKA), stroma-derived candidates versican (VCAN), and syndecan-3 (SDC), which performed significantly better than the recently reported biomarker fibroblast growth factor 18 (FGF18) to discern malignant from benign conditions. Furthermore, elevated MAL and AURKA expression levels correlated significantly with a poor prognosis. We identified promising novel candidates and found the stroma of EOC to be a suitable compartment for biomarker discovery.

Heterogeneous pathway activation and drug response modelled in colorectal-tumor-derived 3D cultures.

Organoid cultures derived from colorectal cancer (CRC) samples are increasingly used as preclinical models for studying tumor biology and the effects of targeted therapies under conditions capturing in vitro the genetic make-up of heterogeneous and even individual neoplasms. While 3D cultures are initiated from surgical specimens comprising multiple cell populations, the impact of tumor heterogeneity on drug effects in organoid cultures has not been addressed systematically. Here we have used a cohort of well-characterized CRC organoids to study the influence of tumor heterogeneity on the activity of the KRAS/MAPK-signaling pathway and the consequences of treatment by inhibitors targeting EGFR and downstream effectors. MAPK signaling, analyzed by targeted proteomics, shows unexpected heterogeneity irrespective of RAS mutations and is associated with variable responses to EGFR inhibition. In addition, we obtained evidence for intratumoral heterogeneity in drug response among parallel "sibling" 3D cultures established from a single KRAS-mutant CRC. Our results imply that separate testing of drug effects in multiple subpopulations may help to elucidate molecular correlates of tumor heterogeneity and to improve therapy response prediction in patients.