Suzan McNamara

Retinoic acid targets DNA-methyltransferases and histone deacetylases during APL blast differentiation in vitro and in vivo.

The acute promyelocytic leukemia (PML)-retinoic acid receptor α (RARα) fusion product recruits histone deacetylase (HDAC) and DNA methyltransferase (DNMT) activities on retinoic acid (RA)-target promoters causing their silencing and differentiation block. RA treatment induces epigenetic modifications at its target loci and restores myeloid differentiation of APL blasts. Using RA-sensitive and RA-resistant APL cell lines and primary blasts, we addressed the functional relevance of the aberrant methylation status at the RA-target promoter RARβ2 and the mechanism by which methylation is reversed by RA. RA decreased DNMT expression and activity, which correlated with demethylation at specific sites on RARβ2 promoter/exon-1, and the ability of APL blasts to differentiate in vitro and in vivo. None of these events occurred in an RA-resistant APL cell line containing a PML-RARα defective for ligand binding. The specific contribution of the HDAC and DNMT pathways to the response of APL cells to RA was also tested by inhibiting these enzymatic activities with TSA and/or 5-azacytidine. In RA-responsive and RA-resistant APL blasts, TSA and 5-azacytidine induced specific changes on the chromatin state at RA-target sites, increased the RA effect on promoter activity, endogenous RA-target gene expression and differentiation. These results extend the rationale for chromatin-targeted treatment in APL and RA-resistant leukemias.

Topoisomerase IIβ Negatively Modulates Retinoic Acid Receptor α Function: a Novel Mechanism of Retinoic Acid Resistance

ABSTRACT Interactions between retinoic acid (RA) receptor α (RARα) and coregulators play a key role in coordinating gene transcription and myeloid differentiation. In patients with acute promyelocytic leukemia (APL), the RARα gene is fused with the promyelocytic leukemia (PML) gene via the t(15;17) translocation, resulting in the expression of a PML/RARα fusion protein. Here, we report that topoisomerase II beta (TopoIIβ) associates with and negatively modulates RARα transcriptional activity and that increased levels of and association with TopoIIβ cause resistance to RA in APL cell lines. Knockdown of TopoIIβ was able to overcome resistance by permitting RA-induced differentiation and increased RA gene expression. Overexpression of TopoIIβ in clones from an RA-sensitive cell line conferred resistance by a reduction in RA-induced expression of target genes and differentiation. Chromatin immunoprecipitation assays indicated that TopoIIβ is bound to an RA response element and that inhibition of TopoIIβ causes hyperacetylation of histone 3 at lysine 9 and activation of transcription. Our results identify a novel mechanism of resistance in APL and provide further insight to the role of TopoIIβ in gene regulation and differentiation.

Expanding the use of retinoids in acute myeloid leukemia: spotlight on bexarotene.

In this issue, Tsai et al. ([1][1]) report on a phase-I clinical trial of bexarotene in 27 non-M3 acute myeloid leukemia (AML). AML is characterized by abnormalities in the myeloid line at various stages of commitment and maturation, leading to an accumulation of granulocyte or monocyte precursors.

Abstract LB-231: Genomic profiling in serial metastatic colorectal tumors identifies copy number alterations and spatio temporal intra-patient heterogeneity profiles associated with clinical response. Q-CROC-01: NCT00984048

Introduction: Colorectal cancer (CRC) is the third leading cause of cancer related deaths primarily due to its resistance to current treatments. Studies aiming at understanding mechanisms of resistance have largely investigated the genomic landscape of primary tumors at diagnosis. However, selective pressures during therapy can lead to the expansion of resistant clones and tumor heterogeneity. This highlights the need to characterize the molecular changes of metastasis over time of treatment and response to decipher tumor evolution and therapeutic resistance mechanisms. Methods: Metastatic liver tissue samples were collected at baseline (pre-biopsies) and at the time of resistance (post-biopsies) in responder and non-responder CRC patients undergoing the same first-line treatment. Paired pre/post biopsies were collected from 14 patients including 4 patients with multiple post-biopsies to assess temporal and spatio-temporal tumor heterogeneity following treatment exposure. Biopsies were profiled using exome and transcriptome sequencing as well as high-density Single-Nucleotide Polymorphism (SNP) array analysis to capture chromosomal anomalies, loss of heterozygosity and copy number (CN) variations. Results: Profiling of 45 samples with both high-density SNP array and exome sequencing revealed 97.4% similarity between both technologies in the identification of genes targeted by copy number changes. Using chemo-naive biopsies, we identified 120 CN gains and 47 CN loss that were significantly associated with patient progression free survival. Integrative analysis with transcriptome data revealed that only 10% of the genomic CN gains and 17% of the CN loss correlated with their gene expression levels. Based on CN variants comparison between paired pre/post treatment samples, we found high temporal intra-patient heterogeneity over time of treatment. Interestingly, we observed a relationship between heterogeneity and tumor response; showing that acquired resistant tumors have the highest temporal variations. Conclusion: This study, using a multi-omic approach to profile serial liver metastatic samples in CRC patients, highlights the genomic changes in tumor composition after treatment exposure and constitutes an innovative approach to identify clinical biomarkers and molecular signatures of resistance. Citation Format: Mathilde Couetoux du Tertre, Maud Marques, Karen Gambaro, Michael Witcher, Benoit Samson, Bernard Lesperance, Yoo-Joung Ko, Richard Dalfen, Eve St-Hilaire, Lucas Sideris, Felix Couture, Sabine Tejpar, Ronald Burkes, Mohammed Harb, Errol Camlioglu, Adrian Gologan, Vincent Pelsser, Andre Constantin, Suzan McNamara, Petr Kavan, Claudia Kleinman, Gerald Batist. Genomic profiling in serial metastatic colorectal tumors identifies copy number alterations and spatio temporal intra-patient heterogeneity profiles associated with clinical response. Q-CROC-01: NCT00984048 [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 LB-231.

Abstract 2795: Integrating multiomics discovery approaches to identify biomarkers of therapeutic resistance in metastatic colorectal cancer through analyses of multiple sequential tumor and liquid biopsies; Qcroc01: NCT00984048

Colorectal cancer (CRC) is the 2nd leading cause of cancer related-death in Canada. Clinical responses of metastatic (m)CRC to first-line treatment range from 35 to 60%, but even responders inevitably develop therapeutic resistance. Studies aiming at understanding mechanisms of resistance have largely investigated primary tumors. However, selective pressures during therapy can lead to the expansion of resistant clones and tumor heterogeneity. This highlights the need to characterize the molecular changes of metastasis and plasma over time of treatment and response to decipher tumor evolution and therapeutic resistance mechanisms. In this multicenter study, 52 tissue samples from liver metastasis were collected at baseline (pre-biopsies) and at the time of resistance (post-biopsies) in responder and non-responder mCRC patients (n=44) undergoing the same standard first-line treatments. Multiple post-biopsies also have been harvested in 4 patients, to allow the assessment of tumor heterogeneity and as well as the evolution of the genomic complexity after treatment exposure. Analyses were carried out across multiple omic platforms to identify resistant signatures and characterize molecular changes during treatment. Biopsies were profiled using exome and transcriptome sequencing as well as high-density SNP array analysis to capture chromosomal anomalies, loss of heterozygosity (LOH) and copy number variations (CNV). Additionally, serial blood samples were collected for proteomic, ctDNA and cytokine analysis. Our preliminary analysis of transcriptomes performed on serial biopsies from a set of 11 patients identified genes consistently overexpressed at resistance. Cytogenetics analysis showed similar genomic profiles of matched pre- and post-biopsies and allowed the establishment of LOH and CNV catalogues of liver metastasis, while exome sequencing revealed cumulative somatic mutations over time of treatment, which suggests subclonal and acquired “driver” mutations of resistance. Plasma-derived ctDNA analysis was performed to investigate the mutational status during treatment and whether they correlate with their relative levels in biopsies. Immune gene expression analysis of a test set of 27 metastases revealed strong clustering of 7 metastases due to overexpression of transcripts related to active immune response, allowing to define novel subgroups of patients based on immune response status. Our study, using a multi-omic strategy and integration of independent molecular platforms to profile liver metastasis samples of responder and non-responder mCRC patients, constitutes an innovative approach to identify clinical biomarkers and molecular signature of resistance, which may enhance individualization of cancer medicine and customized therapy. Citation Format: Karen Gambaro, Maud Marques, Ryan Morin, Claudia Kleinman, Michael Witcher, Simon Turcotte, Benoit Samson, Bernard Lesperance, Yoo-Joung Ko, Richard Dalfen, Eve St-Hilaire, Lucas Sideris, Felix Couture, Sabine Tejpar, Ronald Burkes, Mohammed Harb, Francine Aubin, Thierry Alcindor, Errol Camlioglu, Adriana Aguilar, Mathilde Couetoux du Tertre1, Suzan McNamara, Adrian Gologan, Petr Kavan, Gerald Batist. Integrating multiomics discovery approaches to identify biomarkers of therapeutic resistance in metastatic colorectal cancer through analyses of multiple sequential tumor and liquid biopsies; Qcroc01: NCT00984048 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2795. doi:10.1158/1538-7445.AM2017-2795

Abstract 3888: Molecular profiling of sequential biopsies in patients with metastatic colorectal cancer identifies genomic alterations that evolve during first-line therapy and could have therapeutic implications: A prospective study to identify molecular mechanisms of clinical resistance (QCROC-01: NCT00984048).

Therapeutic resistance remains a major obstacle in metastatic colorectal cancer (mCRC) and biomarkers to guide treatment are essential to improving survival and quality of life in mCRC patients. A biopsy-driven prospective study was designed to identify biomarkers and mechanisms of resistance to a standard first-line therapy in patients with mCRC which could be useful in guiding treatment selection (QCROC-01; NCT00984048). We also hoped to recognize molecular changes over time, or resulting from the selection pressure of treatment, which could have implications for subsequent therapy. This study is ongoing and approved at thirteen sites with one-hundred patients enrolled so far. Patients with mCRC receiving FOLFOX (5-fluorouracil, leucovorin and oxaliplatin) with bevacizumab consented to three needle core tumour biopsies at pre-treatment and at the time of resistance. The rate of both patient and physician acceptance of biopsies has steadily risen with time and experience. Serial bloods were also collected for proteomic analysis and circulating tumor DNA. Twenty-five biopsy samples were profiled using exome sequencing (tumor and germ line), RNAseq, low pass genome sequencing and miRNA analysis. Differential gene expression analysis revealed signatures associated with clinical response and resistance when comparing tumours obtained pre- and post-treatment. We detect changes in variant allele fraction including both depletion and enrichment of individual somatic mutations over the course of treatment, the latter of which may indicate subclonal and acquired “driver” mutations that confer therapeutic resistance. A small number of genes show recurrent evidence for changes in clonal enrichment at the time of relapse across multiple patients. These could also represent therapeutic targets for subsequent therapy for these patients, and as such, represent new treatment opportunities. Our findings provide insights into tumor evolution during first-line chemotherapy of mCRC that may hold clues to optimize current first-line therapeutic decision making and identifies potential target pathways for second-line stratification of patients. This study is part of the Canadian Colorectal Cancer Consortium which is a multi-site collaboration funded by the Terry Fox Research Institute and le fonds de recherche du quebec - sante. Citation Format: Suzan McNamara, Ryan Morin, Mathilde Couetoux du Tertre, Rosemary McCloskey, Rebecca Johnston, Daniel Fornika, Benoit Samson, Bernard Lesperance, Thierry Alcindor, Yoo-Joung Ko, Richard Dalfen, Eve St-Hilaire, Lucas Sideris, Felix Couture, Hans Prenen, Sabine Tejpar, Ronald Burkes, Andre Constantin, Errol Camlioglu, Adriana Aguilar, Adrian Gologan, Benoit Tetu, Celia M. Greenwood, Cyrla Hoffert, Samia Qureshi, Zuanel Diaz, Maud Marques, Micheal Witcher, Therese Gagnon-Kugler, Petr Kavan, Gerald Batist. Molecular profiling of sequential biopsies in patients with metastatic colorectal cancer identifies genomic alterations that evolve during first-line therapy and could have therapeutic implications: A prospective study to identify molecular mechanisms of clinical resistance (QCROC-01: NCT00984048). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3888. doi:10.1158/1538-7445.AM2015-3888

Abstract 5534: Building the organization framework for biopsy-driven translational research: The Quebec Clinical Research Organization in Cancer (Q-CROC) experience

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: The success of personalized medicine in oncology relies on translational research efforts to identify biomarkers that will influence clinical management. The discovery and validation of biomarkers is a concerted effort requiring an organizational framework that is often underestimated. The Quebec Clinical Research Organization in Cancer (Q-CROC) consortium is a multi-disciplinary and multi-institutional group of scientists and clinicians devoted to integrating and enhancing translational and clinical research capacity in Quebec. We describe here the organizational framework driving a multicenter, prospective study to identify biomarkers of clinical resistance to first-line therapy in metastatic colorectal cancer ([NCT00984048][1], Q-CROC-01). Results: The Q-CROC consortium has put in place an organizational infrastructure to support the activities and operations of its translational projects. We identified and addressed several critical issues during the course of the Q-CROC-01 translational project that were also common to our subsequent biomarker-driven trial in lymphoma (Q-CROC-02, [NCT01238692][2]) and breast cancer (Q-CROC-03, [NCT01276899][3]). Examples of these issues include: (i) feasibility and burden of tissue collection at participating sites, (ii) limiting pre-analytical variability in blood and tissue specimens for functional downstream applications, (iii) verification of tumor content on biopsy specimens, (iv) tracking sample flow, (v) integration of clinical data with discovery platforms, and (vi) engaging participation throughout all steps of the project. In part to address the above issues, we established five operational Cores: clinical, biobank, biospecimen processing, bioanalytical and bioinformatic. A further challenge was the integration between these Cores, who for the most part operated in silos. We observed that a critical element to unify all components of the consortium was a scientific project management team, consisting of dedicated individuals regularly interacting with each Core to ensure that objectives were aligned and deliverables were met. This academic framework for translational research may be comparable to that of multicenter clinical trials undertaken by industry, but some challenges, including financial and time constraints, data sharing and IP agreements, and engagement of its members, may be more palpable in the academic setting. Conclusion: Infrastructure science is underestimated and under-reported in translational cancer research and is crucial to the success of any large-scale biomarker discovery effort. Our experience with three multi-institutional biomarker-driven trials is that progress hinges upon the availability of an infrastructure that is not only the sum of its parts but that provides a concrete link between each component. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5534. doi:1538-7445.AM2012-5534 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00984048&atom=%2Fcanres%2F72%2F8_Supplement%2F5534.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01238692&atom=%2Fcanres%2F72%2F8_Supplement%2F5534.atom [3]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01276899&atom=%2Fcanres%2F72%2F8_Supplement%2F5534.atom