Heterogeneity Of Resistance Mechanisms Research Articles

Quantification of cerebrospinal fluid tumor DNA in lung cancer patients with suspected leptomeningeal carcinomatosis

Cerebrospinal fluid tumor-derived DNA (CSF-tDNA) analysis is a promising approach for monitoring the neoplastic processes of the central nervous system. We applied a lung cancer-specific sequencing panel (CAPP-Seq) to 81 CSF, blood, and tissue samples from 24 lung cancer patients who underwent lumbar puncture (LP) for suspected leptomeningeal disease (LMD). A subset of the cohort (N = 12) participated in a prospective trial of osimertinib for refractory LMD in which serial LPs were performed before and during treatment. CSF-tDNA variant allele fractions (VAFs) were significantly higher than plasma circulating tumor DNA (ctDNA) VAFs (median CSF-tDNA, 32.7%; median plasma ctDNA, 1.8%; P < 0.0001). Concentrations of tumor DNA in CSF and plasma were positively correlated (Spearman’s ρ, 0.45; P = 0.03). For LMD diagnosis, cytology was 81.8% sensitive and CSF-tDNA was 91.7% sensitive. CSF-tDNA was also strongly prognostic for overall survival (HR = 7.1; P = 0.02). Among patients with progression on targeted therapy, resistance mutations, such as EGFR T790M and MET amplification, were common in peripheral blood but were rare in time-matched CSF, indicating differences in resistance mechanisms based on the anatomic compartment. In the osimertinib cohort, patients with CNS progression had increased CSF-tDNA VAFs at follow-up LP. Post-osimertinib CSF-tDNA VAF was strongly prognostic for CNS progression (HR = 6.2, P = 0.009). Detection of CSF-tDNA in lung cancer patients with suspected LMD is feasible and may have clinical utility. CSF-tDNA improves the sensitivity of LMD diagnosis, enables improved prognostication, and drives therapeutic strategies that account for spatial heterogeneity in resistance mechanisms.

Abstract IA001: Patient-derived tumor organoids to probe the cancer immune interface

Abstract The development of rational (combination) immunotherapies is limited by an incomplete understanding of what determines a tumor’s sensitivity to immune attack. Despite great heterogeneity in resistance mechanisms between patients, functional model systems to study cancer/immune cell interactions at the level of individual patients have been lacking. Here we highlight the potential of organoid/immune cell co-cultures as individualized models of anti-tumor immunity. First, we established a co-culture platform of autologous tumor organoids and peripheral blood lymphocytes from patients with mismatch-repair deficient colorectal cancer (dMMR-CRC) and non-small-cell lung cancer (NSCLC). This allows enriching tumor-reactive T cells from peripheral blood of individual patients, and creating personalized test systems to evaluate their capacity to kill matched tumor organoids. Second, subclonal immune escape is a major barrier to achieving complete responses to cancer immunotherapies. While most tumours consist of multiple genetically distinct clones, directly investigating the potential for immune escape at the clonal level has been impossible so far, due to challenges in isolating and propagating individual tumour subclones from human cancers for functional studies. By leveraging the multi-region TRACERx study, we established multi-region organoid libraries of &amp;gt;20 parallel sublines from patients with NSCLC. Co-cultures of each subline with autologous tumour infiltrating lymphocytes (TIL) reveal substantial heterogeneity in the capacity of individual clonal sublines to elicit a T-cell response. We show that subclonal immune escape can be mediated by antigen-dependent and -independent mechanisms. These results show (i) that tumour evolution can give rise to distinct cancer clones with intrinsic differences in immune evasion capacity and (ii) provide an approach to prospectively identify and isolate immune evading subclones from cancer patients. Third, analysis of a clinical cohort of patients treated with anti-PD1 therapy revealed that gamma delta T cells are critical mediators of the response in dMMR-CRC patients with genetic inactivation of beta-2-microglobulin (B2M). Co-cultures of organoids and gamma delta T cells provide direct evidence that loss of B2M increases reactivity of patient-derived gamma delta T cells towards tumor organoids. This highlights the need for functional models that move beyond conventional T cells. Future developments are directed towards establishment of complex co-culture systems to study how communication between organoids and immune cells is mediated by cell surface factors and secreted molecules, and how cellular crosstalk is altered in cancer. Citation Format: Krijn K Dijkstra. Patient-derived tumor organoids to probe the cancer immune interface [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA001.

Development of resistance to FGFR inhibition in urothelial carcinoma via multiple pathways in vitro.

Alterations of fibroblast growth factor receptors (FGFRs) are common in bladder and other cancers and result in disrupted signalling via several pathways. Therapeutics that target FGFRs have now entered the clinic, but, in common with many cancer therapies, resistance develops in most cases. To model this, we derived resistant sublines of two FGFR-driven bladder cancer cell lines by long-term culture with the FGFR inhibitor PD173074 and explored mechanisms using expression profiling and whole-exome sequencing. We identified several resistance-associated molecular profiles. These included HRAS mutation in one case and reversible mechanisms resembling a drug-tolerant persister phenotype in others. Upregulated IGF1R expression in one resistant derivative was associated with sensitivity to linsitinib and a profile with upregulation of a YAP/TAZ signature to sensitivity to the YAP inhibitor CA3 in another. However, upregulation of other potential therapeutic targets was not indicative of sensitivity. Overall, the heterogeneity in resistance mechanisms and commonality of the persister state present a considerable challenge for personalised therapy. Nevertheless, the reversibility of resistance may indicate a benefit from treatment interruptions or retreatment following disease relapse in some patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

More to the RAS Story: KRASG12C Inhibition, Resistance Mechanisms, and Moving Beyond KRASG12C.

Despite the discovery of RAS oncogenes in human tumor DNA 40 years ago, the development of effective targeted therapies directed against RAS has lagged behind those more successful advancements in the field of therapeutic tyrosine kinase inhibitors targeting other oncogenes such as EGFR, ALK, and ROS1. The discoveries that (1) malignant RAS oncogenes differ from their wild-type counterparts by only a single amino acid change and (2) covalent inhibition of the cysteine residue at codon 12 of KRASG12C in its inactive GDP-bound state resulted in effective inhibition of oncogenic RAS signaling and have catalyzed a dramatic shift in mindset toward KRAS-driven cancers. Although the development of allele-selective KRASG12C inhibitors has changed a treatment paradigm, the clinical activity of these agents is more modest than tyrosine kinase inhibitors targeting other oncogene-driven cancers. Heterogeneous resistance mechanisms generally result in the restoration of RAS/mitogen-activated protein kinase pathway signaling. Many approaches are being evaluated to overcome this resistance, with many combinatorial clinical trials ongoing. Furthermore, because KRASG12D and KRASG12V are more prevalent than KRASG12C, there remains an unmet need for additional therapeutic strategies for these patients. Thus, our current translational standing could be described as "the end of the beginning," with additional discovery and research innovation needed to address the enormous disease burden imposed by RAS-mutant cancers. Here, we describe the development of KRASG12C inhibitors, the challenges of resistance to these inhibitors, strategies to mitigate that resistance, and new approaches being taken to address other RAS-mutant cancers.

Abstract 3244: Exploring the mechanisms of olaparib resistance in resistant-BRCA1 deficient murine ovarian cells

Abstract Background: High-grade serous ovarian carcinoma (HGSOC) is the most frequent histotype of ovarian cancer. More than half of HGSOC is characterized by homologous recombination deficiency (HRD) due to mutations in genes involved in this pathway, including BRCA1/2. Olaparib is a poly(ADP-ribose) polymerase inhibitor (PARPi) recently approved in front line and maintenance therapy in platinum-sensitive BRCA mutated ovarian cancer patients. Despite olaparib clinical benefits, this treatment is associated with the development of resistance. A better understanding of the mechanisms at its basis could help in finding strategies to delay or overcome/counteract it, possibly translatable in the clinical setting. Methods: We generated an olaparib resistant cell line (BRCA1-/- OlaR), starting from the BRCA1 and TP53 deleted murine ID8 cells (BRCA1-/-) by step wise increasing drug concentrations. Sensitive and resistant cell lines were biologically and pharmacologically characterized and the molecular mechanisms at the basis of olaparib resistance investigated. Cellular viability was assessed by MTS assay, for molecular studies real-time PCR, western blot and RAD51-foci immunofluorescence were performed. Results: BRCA1-/- OlaR cells were 24 fold time more resistant than BRCA1-/- cells and displayed a similar in vitro cell growth compared to the parental cells. Olaparib treatment at IC50 dose caused a longer and stronger G2-M block of the cell cycle in sensitive as compared to resistant cells. BRCA1-/- OlaR cells were cross-resistant to the other PARPis tested, suggesting a common mechanism of resistance. Platinum compounds showed similar cytotoxic activity in sensitive and resistant cells, while a partial cross-resistance was observed with doxorubicin. Interestingly, resistant cells were less responsive to a panel of DNA damage response (DDR) inhibitors (i.e. ATR, WEE1 and CHK1 inhibitors) compared to parental ones; however their combinations with olaparib were synergic in both the sensitive and resistant lines. A partial restoration of the HR pathway (increase in RAD51 foci formation after olaparib and IR treatment), a downregulation of PARP1 protein levels without mutation in PARP1 gene and an upregulation of the MDR transcript could be found in BRCA1-/- resistant cells, strongly supporting multiple heterogeneous mechanisms of resistance. Conclusions: The HR deficient cell line resistant to olaparib we generated showed multiple mechanisms of resistance. The combinations of olaparib with different DDR inhibitors were equally active in sensitive and resistant cells. Citation Format: Michela Chiappa, Martina Anselmi, Luca Russo, Monica Lupi, Nicolò Panini, Federica Guffanti, Giovanna Damia. Exploring the mechanisms of olaparib resistance in resistant-BRCA1 deficient murine ovarian cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3244.

Abstract PD2-01: A platform of CDK4/6 inhibitor-resistant patient-derived breast cancer organoids illuminates mechanisms of resistance and therapeutic vulnerabilities

Abstract CDK4/6 inhibitors (CDK4/6i) in combination with antiestrogens have revolutionized the treatment of ER+ metastatic breast cancer (MBC), significantly prolonging survival. However, this combination is not curative, and tumors eventually acquire resistance. Following progression on this combination, patients are left with limited treatment options. A diverse array of mechanisms of resistance to CDK4/6i + antiestrogens have been described. However, laboratory models that capture this heterogeneity of resistance mechanisms are lacking. Patient-derived organoids (PDOs) provide a rapid, robust and reliable platform that recapitulates intra-tumor heterogeneity, partially mimics the cancer microenvironment, and accurately predicts drug response. We aspired to generate a platform of CDK4/6i-resistant breast cancer PDOs to serve as models for understanding acquired resistance to CDK4/6i + antiestrogens and identifying therapies to overcome resistance. We successfully established 16 PDOs out of 32 biopsies (50% efficiency) of metastates from patients with ER+ MBC progressing on CDK4/6i (palbociclib or abemaciclib) + antiestrogens (letrozole or fulvestrant; median response to combination = 9 months). Our collection includes PDOs derived from lobular (n=3) and inflammatory (n=2) breast cancers and reflects racial/ethnic diversity (50% white/not Hispanic; 18.8% Hispanic; 12.5% Black; 12.5% other/unknown). Next-gen sequencing reports were available for 10 patients from which organoids were established, revealing alterations associated with CDK4/6i and/or antiestrogen resistance, including ESR1 (n=2), HER2/ERBB2 (n=2), PTEN (n=2), CCNE1 (n=1), NF1 (n=1), and ARID1A (n=1). Furthermore, one biopsy and its derived organoid lost ER expression, and 5 harbored PIK3CA activating mutations. Thus far, we have performed targeted DNA-sequencing on 7 PDOs and found 13/15 (86.7%) concordance with driver mutations from tumor NGS reports. PDOs established from CDK4/6i-resistant biopsies maintained resistance to palbociclib or abemaciclib ± fulvestrant (500 nM each) in 3D cell viability assays (6 days of treatment). In contrast, control PDOs established from primary ER+ breast cancer surgical samples (n=2) were sensitive to each CDK4/6i ± fulvestrant (median viability for combination=25.6-31.5% for control vs 65.2-80.5% for resistant). GSEA analysis of RNA-seq data from control (n=2) and CDK4/6i-resistant (n=6) PDOs cultured in estrogen-depleted media ± 200 nM palbociclib revealed that palbociclib treatment resulted in downregulation of E2F target and G2M checkpoint signatures in control but not resistant PDOs. Next, we performed a high-throughput screen of 1,000 compounds in 3 resistant PDOs. One PDO showed exquisite sensitivity to G2/M cell cycle checkpoint components, including CDK1, PLK1, Aurora kinase, ATR, Chk1, and Wee1 inhibitors. Finally, treatment of 10 resistant PDOs with the CDK2/4/6 inhibitor PF-06873600 revealed that the CCNE1 (cyclin E1)-amplified PDO was highly sensitive (IC50=130 nM vs &amp;gt;1000 nM), supporting that CCNE1-amplified tumors are vulnerable to CDK2 inhibition. Conclusions: PDOs can be successfully established from ER+ MBC biopsies, maintain the resistant phenotype in culture, retain driver alterations found in tumors from which they were derived, and fail to suppress E2F targets following treatment with CDK4/6i. Therefore, these PDOs represent valuable models to understand and explore diverse mechanisms of CDK4/6i resistance and therapeutic vulnerabilities. Citation Format: Ariella B. Hanker, Sumanta Chatterjee, Yunguan Wang, Dan Ye, Dhivya R. Sudhan, Brian M. Larsen, Lauren C. Smith, Yilin Zhang, Vishal Kandagatla, Kuntal Majmudar, Ezequiel Renzulli, Saurabh Mendiratta, Kimberly Blackwell, Alana L. Welm, Sunati Sahoo, Nisha Unni, Cheryl M. Lewis, Tao Wang, Ameen A. Salahudeen, Carlos L. Arteaga. A platform of CDK4/6 inhibitor-resistant patient-derived breast cancer organoids illuminates mechanisms of resistance and therapeutic vulnerabilities [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD2-01.

Gram-Negative Bacteria Bloodstream Infections in Patients with Hematological Malignancies - The Impact of Pathogen Type and Patterns of Antibiotic Resistance: A Retrospective Cohort Study.

BackgroundEnterobacteriaceae (EB) and non-fermentative bacteria (NFB) are the main pathogens responsible for gram-negative bloodstream infections (GN-BSI) in patients with hematological malignancies (HMs). These two pathogen types have heterogeneous resistance mechanisms to antibiotics. However, the impact of pathogen species and pattern of antibiotic resistance on the outcomes of patients with HMs remains unclear.MethodsWe retrospectively collected clinical data of patients with HMs at three comprehensive hospitals in Hunan Province, China, between January 2010 and May 2018. The data analyzed the impact that different species and patterns of antibiotic resistance had on the outcome of patients with HMs.ResultsThe majority of the 835 monomicrobial isolates collected from patients with HMs and GN-BSIs were Enterobacteriaceae (75.7%). While detections of MDR pathogens in BSIs as a whole are decreasing, sub-analysis shows that detections of extended spectrum β-lactamase-producing (ESBL) Enterobacteriaceae and carbapenem-resistant pathogens in BISs are rising. Comparing different species, the early mortality rate associated with infections caused by NFB was significantly higher than infections caused by Enterobacteriaceae (22.6% vs 9.7%, p < 0.001). Across different multidrug-resistant (MDR) patterns, ESBL bacteria did not have a significant impact on health outcomes. Carbapenem-resistant bacteria, on the other hand, were observed to significantly affect early mortality rate, such as carbapenem-resistant Klebsiella pneumoniae (36.0% vs 7.6%, P < 0.001) and carbapenem-resistant non-fermentative bacteria (44.7% vs 16.5%, P < 0.001).ConclusionOur findings suggest that both species and patterns of antibiotic resistance can affect the early mortality of patients with HMs during BSI.

Abstract 598: Resistance mechanisms to BRAF inhibition identified by single circulating tumor cell and cell-free tumor DNA molecular profiling in BRAF-mutant non-small cell lung cancer

Abstract Background: Combination therapy with dabrafenib + trametinib demonstrated robust activity in patients (pts) with BRAFV600E-mutant advanced non-small cell lung cancer (NSCLC), but its resistance mechanisms are poorly known. Liquid biopsy components such as circulating tumor cells (CTCs) and cell-free (cf) tumor DNA can provide a comprehensive genomic picture of tumor content. Molecular profiling of single CTCs from pts with BRAF-V600Emutant NSCLC was performed to carry out a pilot study to identify resistance mutations at failure to dabrafenib + trametinib and to compare the mutations detected on CTCs to the mutations found on cfDNA and tumor biopsies. Patients and Methods: Eight pts with advanced BRAFV600E-mutant NSCLC at failure to dabrafenib + trametinib were prospectively enrolled between Jul 2018 and Mar 2019 at Gustave Roussy (IDRCB2008-A00585-50). Bloods samples were collected. Matched tissue-cfDNA and CTCs were available in 3 pts and matched tissue-CTCs for 4 pts. Single CTC isolation strategy included RosetteSep enrichment, immunofluorescent staining (Hoechst/CD45/cytokeratins) and fluorescence activated cell-sorting. The process to identify CTC mutations included Ampli1 whole-genome amplification, quality controls, multiplex targeted PCR with the Ampli1 CHPCustomBeta cancer panel developed by (Menarini Silicon Biosystems) and next-generation sequencing (NGS). The cfDNA was analyzed using InVisionFirst-Lung. Tissue samples were analyzed using targeted NGS in the MATCH-R trial (Recondo G; NPJ Precis Oncol 2020). Results: Single CTCs were isolated from 7 pts. As baseline characteristics, the median age was 66 years, 5 (71%) were smoker; all the pts with adenocarcinoma histology. Most of the pts received dabrafenib + trametinib as 2nd line (86%). The median of CTCs isolated by patient was 20 (8-28). A wide spectrum of mutations in CTCs was observed at treatment failure that were involved in the main cancer pathways, including MAPK (n=1; NRAS), tyrosine kinase receptors (n=5; EGFR, ALK, FLT3, HER2,…), signal transduction (n=4; IDH1, EZH2,⋯), and DNA repair (n=2; AKT1, ATM,⋯). In the same CTC, several mutations were observed in 5/7 patients, commonly involving more than one cancer pathways. A higher degree of mutational diversity was observed in CTCs compared to tumor tissue biopsies and cfDNA. In the 3 patients with an available tumor/liquid biopsy, only 1 shared mutations between CTCs and matched tumor and cfDNA. Conclusion: Single CTC profiling reveals a wide spectrum of therapeutic resistance mutations not detected by other analyses in pts with BRAFV600E-mutant NSCLC at failure to dabrafenib + trametinib. Integration of single CTC sequencing to tumor and cfDNA analysis, provides important perspectives to assess heterogeneous resistance mechanisms and to guide precision medicine in BRAFV600E- NSCLC. Citation Format: Laura Mezquita, Marianne Oulhen, Agathe Aberlenc, Marc Deloger, Aurélie Honoré, Marianna Garonzi, Genny Buson, Claudio Forcato, Yann Lecluse, Mihaela Aldea, Maud NgoCamus, Claudio Nicotra, Karen Howarth, Ludovic Lacroix, Luc Friboulet, Benjamin Besse, Nicolò Manaresi, David Planchard, Françoise Farace. Resistance mechanisms to BRAF inhibition identified by single circulating tumor cell and cell-free tumor DNA molecular profiling in BRAF-mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 598.

Abstract 555: Longitudinal exome-scale liquid biopsy monitoring of evolving therapeutic resistance mechanisms in head and neck squamous cell carcinoma patients receiving anti-PD-1 therapy

Abstract Background Typical liquid biopsy panels capture a relatively small number of variants, and likely under-represent the heterogeneity of resistance in late-stage cancers. This reduced scope can result in overlooked therapeutic biomarkers which respond dynamically to treatment, as well as potentially missed resistance mechanisms and pathway-level events. To address the challenges associated with identifying multiple concurrent heterogeneous resistance mechanisms in individual patients, we evaluated longitudinal whole exome sequencing of cell free DNA (cfDNA) and solid tumor biopsies from head and neck squamous cell carcinoma (HNSCC) patients that received anti-PD1 therapy. Using this approach, we identified evolving variant and pathway-level resistance mechanisms in cfDNA, as a complement to tumor biopsy derived information, and identified differences in putative neoantigens found in tissue and cfDNA. Methods Pre- and post-intervention matched tumor, normal and plasma samples were obtained from a pilot cohort of 13 patients with HNSCC. Following baseline sample collection, all patients received a single dose of nivolumab. The primary tumor was then resected, approximately one month later when possible, or a second biopsy was collected where resection was impractical. Paired tumor and normal samples were profiled using ImmunoID NeXTTM, an augmented exome/transcriptome platform and analysis pipeline. Exome-scale cfDNA profiling of matched plasma samples was performed using the NeXT Liquid BiopsyTM platform to detect somatic variants. Data from these two platforms were then compared with corresponding clinical findings. Results We observed a rapid evolution of the tumor microenvironment and disease mutation profile following therapy, with strong concordance detected between plasma and tumor variants at each timepoint. Post-therapy interrogation of cfDNA revealed dynamic changes in numerous oncogenes and clinically relevant pathways, such as ERK1/2 and MAPK, that were not observed in solid tumor. These findings suggest that single-lesion biopsy of the primary tumor misses co-occurring, clinically relevant resistance alterations. Median post-treatment neoantigen count was reduced in solid tumor, but increased in cfDNA. HLA-specific loss of heterozygosity (LOH) was identified in a number of subjects, likely resulting in reduced neoepitope presentation in those cases. Conclusions Exome-wide somatic events were reliably detected in cfDNA, providing additional potential biomarkers to complement those identified in solid tumor. As we increase our cohort size, we expect that identification of biomarkers from both exome scale tissue biopsy and cfDNA will provide a more comprehensive view into therapeutic response and resistance mechanisms in HNSCC patients missed with typical liquid biopsy panels. Citation Format: Charles W. Abbott, Nikita Bedi, Simo V. Zhang, Josette Northcott, Robin LI, Rachel Marty Pyke, Eric Levy, Rebecca Chernock, Mena Mansour, A. Dimitrios Colevas, John Lyle, John B. Sunwoo, Sean Boyle, Richard Chen. Longitudinal exome-scale liquid biopsy monitoring of evolving therapeutic resistance mechanisms in head and neck squamous cell carcinoma patients receiving anti-PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 555.

BCL2A1: A Novel Target in Refractory Acute Myeloid Leukemia with FLT3-ITD/D835 Dual Mutations

Internal tandem duplications in the juxtamembrane domain of FMS-like tyrosine kinase 3 gene (FLT3-ITD) and missense mutations in the gene's tyrosine-kinase domain (FLT3-TKD) play critical roles in the pathophysiology of acute myeloid leukemia (AML). Recent study revealed that AML cells resistant to quizartinib, a type II TKI, consist of heterogeneous clonal populations harboring wild-type FLT3 as well as FLT3-ITD, FLT3-TKD and FLT3-ITD/TKD mutations, and on- and off-target mechanisms may contribute to the resistance to TKIs. To overcome the heterogeneous resistance mechanisms of FLT3-ITD and TKD mutations, various combinatorial therapies have been investigated. For example, BCL-2 inhibition in the presence of TKIs increased survival a murine FLT3-ITD AML model, and a phase Ib/II clinical trial of a combination of quizartinib with venetoclax, a BCL2 inhibitor is ongoing in relapsed/refractory FLT3-mutant AML patients (NCT03735875). To dissect underlying mechanisms of drug-resistance and exploring new targets in refractory AML with FLT3-ITD and TKD mutations, we investigated alterations of transcriptome signatures by analyzing AML samples with FLT3-ITD/D835 dual mutations. Previously, we reported BCL2A1 transcriptomes were upregulated in primary AML cells with FLT3-ITD/D835 dual mutations compared to cells with FLT3-ITD mutations only. This was recapitulated in the MV4-11 cells harboring FLT3-ITD/D835 dual mutations after 6 month-exposure to quizartinib. The MV4-11 cells with the FLT3-ITD/D835 dual mutations became resistant to quizartinib, and the cells also became resistant to venetoclax, a BCL2 inhibitor (Yamatani et al. ASH 2019). In this study, we further investigated BCL2A1 as new target in refractory AML with FLT3-ITD/D835 dual mutations. First, we examined whether overexpression of BCL2A1 induces drug-resistance in MV4-11 and Molm13 cell lines with FLT3-ITD. While parental MV4-11 and Molm13 cells are sensitive to venetoclax and quizartinib, MV4-11 and Molm13 cells transfected with lentivirus carrying BCL2A1 became resistant to venetoclax (IC50: MV4-11 with BCL2A1 over-expression &amp;gt;1000 nM vs. mock vector 0.71 nM; Molm13 with over-expression &amp;gt;1000 nM vs. mock vector 0.38 nM, 72h). In contrast, the sensitivity to quizartinib was retained in the BCL2A1 overexpressing MV4-11 and Molm13 cells. These findings indicate that the overexpression of BCL2A1 could play a role in the acquired resistance to venetoclax, but not to quizartinib. Bromodomain-containing protein 4 (BRD4), a family member of bromodomain and extra-terminal motif (BET) is known to transcriptionally modulate BCL2A1 gene expression. Next, we examined effects of CPI-0610, a BET inhibitor, on MV4-11 cells with FLT3-ITD or the FLT3-ITD/D835 dual mutation. CPI-0610 inhibited cell growth of MV4-11 cells by inducing apoptosis irrespective of co-existing FLT3 mutations (IC50: FLT3-ITD/D835, 255 nM vs. FLT3-ITD, 191 nM, 72h). Immunoblotting analyses confirmed that BET inhibition by CPT-0610 decreased the expression of BCL2A1 in MV4-11 cells FLT3-ITD/D835. WIn conclusion, transcriptome analysis and molecular pharmacological approaches identified alterations in the anti-apoptotic BCL2 family proteins in double-mutant FLT3 leukemias. BCL2A1 upregulation might be involved in drug resistance of FLT3-ITD/D835 dual mutant AML cells, and could be a promising new target in refractory AML with FLT3-ITD/D835 dual mutations. Disclosures Konopleva: Cellectis: Research Funding; Eli Lilly: Research Funding; Calithera: Research Funding; Sanofi: Research Funding; Ascentage: Research Funding; AstraZeneca: Research Funding; Agios: Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Kisoji: Consultancy; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents &amp; Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Rafael Pharmaceutical: Research Funding; Ablynx: Research Funding; Amgen: Consultancy. Carter:Syndax: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding; Ascentage: Research Funding. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research &amp; Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

Spatial heterogeneity of acquired resistance mechanisms to 1st/2nd generation EGFR tyrosine kinase inhibitors in lung cancer

BackgroundOvercoming acquired resistance against targeted therapies to improve outcomes of lung cancer patients harboring driver mutations is a critical issue. While drug therapy oriented to a resistance mechanism appears attractive, spatial heterogeneity of resistance mechanisms in each patient will diminish treatment efficacy. However, the frequency, clinical backgrounds, clinical implications, and patterns of spatial heterogeneity in resistance mechanisms to EGFR tyrosine kinase inhibitors (TKIs) are largely unknown. Patients and methodsThis study included 128 specimens from 24 autopsied patients with lung adenocarcinoma harboring EGFR mutation. Acquired resistance mechanisms reported as relatively frequent in lung cancer, e.g., T790 M and other secondary EGFR mutations, MET and ERBB2 gene amplification, and histological transformation, were retrospectively examined. All patients had received 1st/2nd generation EGFR-TKI and showed acquired resistance to the drug before death. No patient received osimertinib. ResultsNo resistance mechanism was identified in two patients. T790M mutation was detected in 20 patients (83 %); however, nine of these patients also had lesions without T790M mutation. Among 22 patients whose resistance mechanisms were identified, ten had spatial heterogeneity of resistance mechanisms (45 %), and these patients had significantly shorter time-to-treatment failure compared with those without heterogeneity (median 4.7 months vs. 14.7 months, p = 0.0004). ConclusionWe observed significant spatial heterogeneity of acquired resistance mechanisms to EGFR-TKIs in lung adenocarcinoma. Our results also indicate that the incidence of resistance mechanisms may vary based on the biopsied tumor locations.

EGFR tyrosine kinase inhibitor therapy continuation with high-dose hypofractionated radiotherapy in EGFR-mutated non-small cell lung cancer (NSCLC) patients with oligoprogressive disease.

e21580 Background: EGFR tyrosine kinase inhibitors (TKIs) represent the standard first-line therapy for advanced non-small cell lung cancer (NSCLC) patients with activating EGFR mutations. However, despite initial marked responses, tumors invariably develop acquired resistance to TKIs. Oligoprogression is commonly observed during treatment with oncogene-directed therapies, including EGFR TKIs, and refers to patients who experience disease progression only in limited sites as a result of heterogeneous mechanisms of resistance. The use of local ablative treatments for these resistant lesions may extend the duration of TKI therapy and potentially improve long-term disease control and survival. We e retrospectively analyzed the efficacy of EGFR TKI therapy continuation with high-dose hypofractionated radiation therapy (RT), in EGFR-mutant NSCLC patients with oligoprogressive disease. Methods: Patients with metastatic EGFR mutant NSCLC who developed oligoprogression during first-line treatment with gefitinib were included in this analysis. We evaluated progression-free survival 1 (PFS 1), defined as the time from initiation of TKI therapy until development of oligoprogression or death, and PFS 2, defined as time of focal progression until further progression of disease or death. Overall survival and safety were also assessed. Results: Thirty-six patients were included in the study. The median PFS 1 was 12.5 (4.0-23.2) months. High-dose hypofractionated RT consisted of intensity-modulated RT in 23 patients (64%) and stereotactic radiotherapy in 13 cases (36%). The median PFS 2 was 6.3 (2-12.5) months. Overall survival was 38.7 months (9.0-46.3). The treatment was well-tolerated and no patient had to discontinue TKI therapy because of adverse events during radiotherapy. Conclusions: Our therapeutic strategy, including high-dose hypofractionated RT in addition to TKI therapy, was feasible in the clinical setting and was associated with significant prolongation of disease control and improvement of survival outcomes, while being associated with manageable side effects. Our study further support the use of definitive therapeutic approaches in oligoprogressive disease, especially in oncogene-driven tumors. Molecular profiling of metastatic sites remains crucial to identify novel biomarkers, involved in the development of acquired resistance and oligoprogression, that may be useful to select patients for local treatments.

Acquired resistance to osimertinib in patients with non-small-cell lung cancer: mechanisms and clinical outcomes

PurposeOsimertinib, a third-generation epidermal growth factor receptor tyrosine-kinase inhibitor (EGFR-TKI), has demonstrated substantial clinical benefit in patients with non-small-cell lung cancer (NSCLC) who were resistant to early-generation EGFR-TKIs and had acquired a T790M mutation. The aim of our study was to identify the mechanisms underlying resistance to osimertinib and to correlate them with clinical outcomes.MethodsWe retrospectively analyzed patients with advanced NSCLC who received osimertinib for T790M-mutated acquired resistance to prior EGFR-TKIs between March 1, 2017 and December 31, 2018. Patients with paired molecular data of pre-osimertinib and after resistance development, which were not confirmed with small-cell lung cancer (SCLC) transformation, were included in the molecular analysis set.ResultsOf 49 patients evaluated in the molecular analysis set, 24 patients maintained T790M mutation, while 25 patients exhibited T790M-loss. Molecular modifications were identified in 27 of 49 patients including EGFR acquired mutations (C797S, C796S, G796S, V802I, V834L, E758D and G724S), non-EGFR-dependent mutations (PIK3CA, ALK, BRAF, KRAS and TP53), EGFR amplification and MET amplification. At data cutoff, median progression-free survival (PFS) was 9.3 months in the T790M-retain group compared with 7.8 months in T790M-loss patients (P = 0.053). Median PFS was significantly longer in patients with EGFR-dependent resistance mechanism (13.5 months) than in those with alternative pathway activation (8.2 months; P = 0.012).ConclusionsThe study revealed heterogeneous mechanisms of resistance to osimertinib in advanced NSCLC patients and their association with clinical outcomes. Patients who maintained T790M mutation or with EGFR-dependent resistance mechanism had longer clinical outcome benefits.

Loss of CHD1 Promotes Heterogeneous Mechanisms of Resistance to AR-Targeted Therapy via Chromatin Dysregulation

SummaryMetastatic prostate cancer is characterized by recurrent genomic copy number alterations that are presumed to contribute to resistance to hormone therapy. We identified CHD1 loss as a cause of antiandrogen resistance in an in vivo small hairpin RNA (shRNA) screen of 730 genes deleted in prostate cancer. ATAC-seq and RNA-seq analyses showed that CHD1 loss resulted in global changes in open and closed chromatin with associated transcriptomic changes. Integrative analysis of this data, together with CRISPR-based functional screening, identified four transcription factors (NR3C1, POU3F2, NR2F1, and TBX2) that contribute to antiandrogen resistance, with associated activation of non-luminal lineage programs. Thus, CHD1 loss results in chromatin dysregulation, thereby establishing a state of transcriptional plasticity that enables the emergence of antiandrogen resistance through heterogeneous mechanisms.

Acquired Resistance Mutations to ALK Inhibitors Identified by Single Circulating Tumor Cell Sequencing in ALK-Rearranged Non-Small-Cell Lung Cancer.

Patients with anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung cancer (NSCLC) inevitably develop resistance to ALK inhibitors. New diagnostic strategies are needed to assess resistance mechanisms and provide patients with the most effective therapy. We asked whether single circulating tumor cell (CTC) sequencing can inform on resistance mutations to ALK inhibitors and underlying tumor heterogeneity in ALK-rearranged NSCLC. Resistance mutations were investigated in CTCs isolated at the single-cell level from patients at disease progression on crizotinib (n = 14) or lorlatinib (n = 3). Three strategies including filter laser-capture microdissection, fluorescence activated cell sorting, and the DEPArray were used. One hundred twenty-six CTC pools and 56 single CTCs were isolated and sequenced. Hotspot regions over 48 cancer-related genes and 14 ALK mutations were examined to identify ALK-independent and ALK-dependent resistance mechanisms. Multiple mutations in various genes in ALK-independent pathways were predominantly identified in CTCs of crizotinib-resistant patients. The RTK-KRAS (EGFR, KRAS, BRAF genes) and TP53 pathways were recurrently mutated. In one lorlatinib-resistant patient, two single CTCs out of 12 harbored ALK compound mutations. CTC-1 harbored the ALK G1202R/F1174C compound mutation virtually similar to ALK G1202R/F1174L present in the corresponding tumor biopsy. CTC-10 harbored a second ALK G1202R/T1151M compound mutation not detected in the tumor biopsy. By copy-number analysis, CTC-1 and the tumor biopsy had similar profiles, whereas CTC-10 harbored multiple copy-number alterations and whole-genome duplication. Our results highlight the genetic heterogeneity and clinical utility of CTCs to identify therapeutic resistance mutations in ALK-rearranged patients. Single CTC sequencing may be a unique tool to assess heterogeneous resistance mechanisms and help clinicians for treatment personalization and resistance options to ALK-targeted therapies.

LBA8 - Mechanisms of acquired resistance to first-line osimertinib: Preliminary data from the phase III FLAURA study

Background: In the Phase III FLAURA study (NCT02296125), osimertinib showed superior efficacy compared with standard of care (SoC) EGFR-TKIs in patients (pts) with previously untreated EGFRm advanced NSCLC. Here, we report preliminary data on mechanisms of acquired resistance to osimertinib in pts who progressed during the FLAURA study. Methods: Pts with previously untreated EGFRm (tissue, ex19del/L858R) advanced NSCLC (N = 556) were randomised 1:1 to osimertinib 80 mg once daily (QD; n = 279) or SoC EGFR-TKI (n = 277, gefitinib 250 mg QD or erlotinib 150 mg QD). Paired plasma samples were collected at baseline and following RECIST progression and/or treatment discontinuation up to March 2018. Plasma samples were analysed using next generation sequencing (NGS, Guardant Health; Guardant360 73 gene panel or Omni 500 gene panel). Results: In the osimertinib and SoC EGFR-TKI arms, respectively, 113/279 (41%) and 159/277 (57%) pts had experienced a progression event and/or discontinued treatment and had paired plasma samples analysed by NGS. Only pts with detectable plasma EGFRm (ex19del/L858R) at baseline were evaluable for this analysis: 91/113 (81%; osimertinib) and 129/159 (81%; SoC). In the osimertinib arm, there was no evidence of acquired EGFR T790M and the most common acquired resistance mechanism detected was MET amplification (amp; 14/91; 15%), followed by EGFR C797S mutation (6/91; 7%); other mechanisms included HER2 amp, PIK3CA and RAS mutations (2–7%). In the SoC arm, the most common resistance mechanisms were T790M mutation (60/129; 47%), MET amp (5/129; 4%) and HER2 amp (3/129; 2%). Conclusions: In this paired sample preliminary analysis of a subpopulation of pts (with detectable baseline plasma EGFRm) who had experienced disease progression and/or discontinued treatment, heterogeneous resistance mechanisms were detected with first-line osimertinib, with MET amplification and EGFR C797S mutation being the most commonly observed. In line with previous analyses, T790M was acquired in approximately 50% of SoC-treated pts, and none of the osimertinib-treated pts; no unexpected resistance mechanisms were observed in osimertinib-treated pts. Exploration into novel acquired mutations is ongoing. Editorial acknowledgement: Ellen Maxwell, PhD, from iMed Comms, funded by AstraZeneca in accordance with Good Publications Practice (GPP3) guidelines (ismpp.org/gpp3). Clinical trial identification: NCT02296125. Legal entity responsible for the study: AstraZeneca. Funding: AstraZeneca. Disclosure: B.C. Cho: Honoraria/advisory boards: AstraZeneca, Roche, Boehringer Ingelheim, Novartis, MSD, Yuhan; Research grants: AstraZeneca, Novartis, Yuhan; Speakers' bureaus: AstraZeneca, Novartis, MSD, Yuhan. C. Zhou: Honoraria: Roche, Eli Lilly, BI, Merck, Hengrui, Qiru. Y. Ohe: Grants/personal fees: AstraZeneca, Ono Pharmaceutical, Bristol-Myers Squibb, Chugai, MSD, Novartis, Kyorin, Dainippon-Sumitomo, Daiichi-Sankyo, Boehringer Ingelheim, Bayer, Lilly, Pfizer, Ignyta, Kissei. F. Imamura: Honoraria and research grants: AstraZeneca. R. Shah: Advisory boards: AstraZeneca. Y. Rukazenkov: Employee, shareholder: AstraZeneca. A. Todd: Employee of AstraZeneca. A. Markovets, J. Chmielecki: Employee, shareholder: AstraZeneca. C. Barrett: Employee, stock holder: AstraZeneca. J.E. Gray: Advisor: AstraZeneca; Research grant: AstraZeneca and Genentech for work not directly related to this project. S.S. Ramalingam: Advisory board membership: Abbvie, Amgen, AstraZeneca, BMS, Celgene, Genentech, Lilly, Merck, Roche, Nektar, Loxo, Takeda. All other authors have declared no conflicts of interest.

Patient-derived Models of Abiraterone- and Enzalutamide-resistant Prostate Cancer Reveal Sensitivity to Ribosome-directed Therapy

BackgroundThe intractability of castration-resistant prostate cancer (CRPC) is exacerbated by tumour heterogeneity, including diverse alterations to the androgen receptor (AR) axis and AR-independent phenotypes. The availability of additional models encompassing this heterogeneity would facilitate the identification of more effective therapies for CRPC. ObjectiveTo discover therapeutic strategies by exploiting patient-derived models that exemplify the heterogeneity of CRPC. Design, setting, and participantsFour new patient-derived xenografts (PDXs) were established from independent metastases of two patients and characterised using integrative genomics. A panel of rationally selected drugs was tested using an innovative ex vivo PDX culture system. InterventionThe following drugs were evaluated: AR signalling inhibitors (enzalutamide and galeterone), a PARP inhibitor (talazoparib), a chemotherapeutic (cisplatin), a CDK4/6 inhibitor (ribociclib), bromodomain and extraterminal (BET) protein inhibitors (iBET151 and JQ1), and inhibitors of ribosome biogenesis/function (RNA polymerase I inhibitor CX-5461 and pan-PIM kinase inhibitor CX-6258). Outcome measurements and statistical analysisDrug efficacy in ex vivo cultures of PDX tissues was evaluated using immunohistochemistry for Ki67 and cleaved caspase-3 levels. Candidate drugs were also tested for antitumour efficacy in vivo, with tumour volume being the primary endpoint. Two-tailed t tests were used to compare drug and control treatments. Results and limitationsIntegrative genomics revealed that the new PDXs exhibited heterogeneous mechanisms of resistance, including known and novel AR mutations, genomic structural rearrangements of the AR gene, and a neuroendocrine-like AR-null phenotype. Despite their heterogeneity, all models were sensitive to the combination of ribosome-targeting agents CX-5461 and CX-6258. ConclusionsThis study demonstrates that ribosome-targeting drugs may be effective against diverse CRPC subtypes including AR-null disease, and highlights the potential of contemporary patient-derived models to prioritise treatment strategies for clinical translation. Patient summaryDiverse types of therapy-resistant prostate cancers are sensitive to a new combination of drugs that inhibit protein synthesis pathways in cancer cells.

Abstract 937: Longitudinal circulating-tumor DNA profiling of EGFR-mutated non-small cell lung cancer patients treated with EGFR-tyrosine kinase inhibitors

Abstract Background: In EGFR-mutant non-small cell lung cancer (NSCLC), progression disease (PD) under 1st-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) is driven by the EGFR T790M mutation in about 50% of cases. This mutation is targeted with osimertinib, a specific and potent inhibitor that also showed superior efficacy to that of 1st-generation EGFR-TKIs in the 1st-line treatment EGFR+ NSCLC. Strategy: We performed a longitudinal circulating-tumor DNA (ctDNA) analysis to evaluate 1) the dynamics of sensitizing or resistant mutations over time and 2) the genomic alterations associated with EGFR-TKI resistance, in NSCLC patients treated with either 1st-generation EGFR-TKIs (n=24) or with osimertinib (n=20). ctDNA sequencing was performed using InVisionSeq™, an amplicon-based NGS (36-gene panel) assay. Results: Sensitivity was 80% and 100% for the detection of EGFR-activating mutations and the EGFR T790M mutation, respectively, at PD in ctDNA vs tissue biopsies. The type of progression appears to influence the detection of EGFR mutations in plasma. Patients (pts) with exclusively brain or thoracic PD had significantly lower allelic fractions (AFs) of EGFR-activating mutations than pts with systemic PD (P=0.006). AFs of both EGFR-activating and EGFR T790M mutations were highest in patients with progressive metastases in liver or bone. High cell-free DNA load was associated with the presence of TP53 mutations regardless of the time under treatment (n=248 samples). The total number of mutations detected in ctDNA was higher in pts treated with osimertinib vs. 1st-generation EGFR-TKIs (P=0.004), suggesting a potential increase of tumor heterogeneity over time. We observed that the AFs of mutant ctDNA were correlated with response to treatment. Complete clearance of EGFR T790M (AF detection limit: 0.01%) was observed in pts who presented a partial response to osimertinib (n=17), of these, EGFR-activating mutations were found in 4/17 pts (AF:0.05-1.03%). PD was evidenced in 36/44 pts. Emergence of mechanisms of resistance, before the confirmation of clinical PD, was evidenced in 5 and 3 pts under erlotinib and osimertinib, respectively. PD to 1st-generation EGFR-TKIs was mainly driven by EGFR T790M (86%), followed by mutations in PIK3CA or PTEN. We detected the emergence of heterogeneous potential mechanisms of resistance to osimertinib in 3/9 cases, including mutations in EGFR, KRAS, BRAF as well as HER2 and MET amplification. We did not identify genomic drivers of resistance in 16/36 pts, despite evidence of clinical PD. These pts had either exclusive brain or thoracic progression. ctDNA WES will be performed in these samples. Conclusion: Our results suggest that ctDNA sequencing might be a complementary, noninvasive tool to monitor response to treatment and heterogeneous mechanisms of resistance in NSCLC pts treated with EGFR inhibitors. Citation Format: Sandra Ortiz-Cuaran, Aurélie Swalduz, Camille Léonce, Solène Marteau, Séverine Martinez, Gilles Clapisson, Virginie Avrillon, Luc Odier, Lionel Falchero, Pierre Fournel, Emma Green, Clive Morris, Maurice Pérol, Pierre Saintingy. Longitudinal circulating-tumor DNA profiling of EGFR-mutated non-small cell lung cancer patients treated with EGFR-tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 937.

2 Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies

IntroductionClinical effectiveness of targeted therapies is often transitory and virtually all patients develop secondary resistance. The heterogeneous mechanisms of resistance and the sub-clonal evolution pattern of cell populations that emerge upon pharmacological selection limit disease control with subsequent lines of therapy. We used colorectal cancer (CRC) to test the hypothesis that ancestral oncogenic event (such as WNT pathway alterations) shared by every cell sub-clone might be better suited as therapeutic targets than heterogeneous events in the branches, as the former remain present in each drug-resistant cell independently from its genetic drift.Material and methodsExome analysis was performed on a panel of CRC cells that became resistant to commonly used targeted agents to uncover genetic alterations acquired under drug selective pressure. Tumour evolution of resistant cell population was then inferred through EXPANDs software. β-catenin activity, cell growth and death were measured upon functional and pharmacological modulation of WNT pathway in resistant cells and preclinical models established from tissues biopsy obtained from CRC patients who relapsed during treatment with targeted therapies.Results and discussionsExome sequencing and phylogenetic tracking unveiled a complex sub-clonal architecture in CRC resistance cell populations, indicating parallel evolution of multiple independent cellular lineages, each associated with distinct genomic landscapes. Functional and pharmacological inhibition of WNT signalling induces cell death in CRC preclinical models, regardless of drug type or resistance mechanisms. Pharmacological blockade of WNT pathway resulted in translocation of β-catenin to the plasma membrane, decreased of β-catenin dependent Tcf/LEF transcriptional activity, and cell growth impairment despite massive molecular drift of resistant derivatives. Concomitant blockade of WNT and MAPK signalling restrained evolution of drug-resistant clones and prevent onset of secondary resistance.ConclusionIn summary, reliance of CRC on the WNT pathway is preserved in later phases of colorectal carcinogenesis when tumours face genomic bottlenecks and sub-clonal evolution driven by administration of target therapies; thus offering the possibility of a common therapeutic strategy to overcome secondary resistance to treatments.

Abstract 205: Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies

Abstract Clinical effectiveness of targeted therapies is often transitory and virtually all patients develop secondary resistance. The heterogeneous mechanisms of resistance and the sub-clonal evolution pattern of cell populations that emerge upon pharmacological selection limit disease control with subsequent lines of therapy. We used colorectal cancer (CRC) to test the hypothesis that ancestral oncogenic event (such as WNT pathway alterations) shared by every cell sub-clone might be better suited as therapeutic targets than heterogeneous events in the branches, as the former remain present in each drug-resistant cell independently from its genetic drift.To test our assumption, we first generated a panel of CRC cells resistant to commonly used targeted agents, including EGFR and BRAF inhibitors. Treatment with targeted therapies, although initially effective, fuels clonal evolution and further amplifies molecular diversity. Exome sequencing and phylogenetic tracking unveiled a complex sub-clonal architecture in CRC resistance cell populations, indicating parallel evolution of multiple independent cellular lineages, associated with distinct genomic landscapes.Functional and pharmacological modulation of WNT signaling induces cell death in CRC preclinical models from patients that relapsed during treatment, regardless of drug type or resistance mechanisms. Pharmacological blockade of WNT ligands secretion resulted in translocation of β-catenin from the cytoplasm and nucleus to the plasma membrane, decreased of β-catenin dependent Tcf/LEF transcriptional activity, and cell growth impairment despite massive molecular drift of resistant derivatives. Concomitant blockade of WNT and MAPK signalling restrained evolution of drug-resistant clones.In summary, reliance of CRC on the WNT pathway is preserved in later phases of colorectal carcinogenesis when tumors face genomic bottlenecks and sub-clonal evolution driven by administration of target therapies; thus offering the possibility of a common therapeutic strategy to overcome secondary resistance to treatments. Citation Format: Mariangela Russo, Simona Lamba, Annalisa Lorenzato, Alberto Sogari, Giorgio Corti, Giuseppe Rospo, Monica Montone, Luca Lazzari, Sabrina Arena, Daniele Oddo, Michael Linnebacher, Andrea Sartore-Bianchi, Filippo Pietrantonio, Salvatore Siena, Federica Di Nicolantonio, Alberto Bardelli. Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 205.