Jelmar Quist

PIM1 kinase regulates cell death, tumor growth and chemotherapy response in triple-negative breast cancer

Triple-negative breast cancers (TNBCs) have poor prognosis and lack targeted therapies. Here we identified increased copy number and expression of the PIM1 proto-oncogene in genomic data sets of patients with TNBC. TNBC cells, but not nonmalignant mammary epithelial cells, were dependent on PIM1 for proliferation and protection from apoptosis. PIM1 knockdown reduced expression of the anti-apoptotic factor BCL2, and dynamic BH3 profiling of apoptotic priming revealed that PIM1 prevents mitochondrial-mediated apoptosis in TNBC cell lines. In TNBC tumors and their cellular models, PIM1 expression was associated with several transcriptional signatures involving the transcription factor MYC, and PIM1 depletion in TNBC cell lines decreased, in a MYC-dependent manner, cell population growth and expression of the MYC target gene MCL1. Treatment with the pan–PIM kinase inhibitor AZD1208 impaired the growth of both cell line and patient-derived xenografts and sensitized them to standard-of-care chemotherapy. This work identifies PIM1 as a malignant-cell-selective target in TNBC and the potential use of PIM1 inhibitors for sensitizing TNBC to chemotherapy-induced apoptotic cell death.

Mouse mammary stem cells express prognostic markers for triple-negative breast cancer

IntroductionTriple-negative breast cancer (TNBC) is a heterogeneous group of tumours in which chemotherapy, the current mainstay of systemic treatment, is often initially beneficial but with a high risk of relapse and metastasis. There is currently no means of predicting which TNBC will relapse. We tested the hypothesis that the biological properties of normal stem cells are re-activated in tumour metastasis and that, therefore, the activation of normal mammary stem cell-associated gene sets in primary TNBC would be highly prognostic for relapse and metastasis.MethodsMammary basal stem and myoepithelial cells were isolated by flow cytometry and tested in low-dose transplant assays. Gene expression microarrays were used to establish expression profiles of the stem and myoepithelial populations; these were compared to each other and to our previously established mammary epithelial gene expression profiles. Stem cell genes were classified by Gene Ontology (GO) analysis and the expression of a subset analysed in the stem cell population at single cell resolution. Activation of stem cell genes was interrogated across different breast cancer cohorts and within specific subtypes and tested for clinical prognostic power.ResultsA set of 323 genes was identified that was expressed significantly more highly in the purified basal stem cells compared to all other cells of the mammary epithelium. A total of 109 out of 323 genes had been associated with stem cell features in at least one other study in addition to our own, providing further support for their involvement in the biology of this cell type. GO analysis demonstrated an enrichment of these genes for an association with cell migration, cytoskeletal regulation and tissue morphogenesis, consistent with a role in invasion and metastasis. Single cell resolution analysis showed that individual cells co-expressed both epithelial- and mesenchymal-associated genes/proteins. Most strikingly, we demonstrated that strong activity of this stem cell gene set in TNBCs identified those tumours most likely to rapidly progress to metastasis.ConclusionsOur findings support the hypothesis that the biological properties of normal stem cells are drivers of metastasis and that these properties can be used to stratify patients with a highly heterogeneous disease such as TNBC.

Do Genetic Susceptibility Variants Associate with Disease Severity in Early Active Rheumatoid Arthritis

Objective. Genetic variants affect both the development and severity of rheumatoid arthritis (RA). Recent studies have expanded the number of RA susceptibility variants. We tested the hypothesis that these associated with disease severity in a clinical trial cohort of patients with early, active RA. Methods. We evaluated 524 patients with RA enrolled in the Combination Anti-Rheumatic Drugs in Early RA (CARDERA) trials. We tested validated susceptibility variants — 69 single-nucleotide polymorphisms (SNP), 15 HLA-DRB1 alleles, and amino acid polymorphisms in 6 HLA molecule positions — for their associations with progression in Larsen scoring, 28-joint Disease Activity Scores, and Health Assessment Questionnaire (HAQ) scores over 2 years using linear mixed-effects and latent growth curve models. Results. HLA variants were associated with joint destruction. The *04:01 SNP (rs660895, p = 0.0003), *04:01 allele (p = 0.0002), and HLA-DRβ1 amino acids histidine at position 13 (p = 0.0005) and valine at position 11 (p = 0.0012) significantly associated with radiological progression. This association was only significant in anticitrullinated protein antibody (ACPA)-positive patients, suggesting that while their effects were not mediated by ACPA, they only predicted joint damage in ACPA-positive RA. Non-HLA variants did not associate with radiograph damage (assessed individually and cumulatively as a weighted genetic risk score). Two SNP — rs11889341 (STAT4, p = 0.0001) and rs653178 (SH2B3-PTPN11, p = 0.0004) — associated with HAQ scores over 6–24 months. Conclusion. HLA susceptibility variants play an important role in determining radiological progression in early, active ACPA-positive RA. Genome-wide and HLA-wide analyses across large populations are required to better characterize the genetic architecture of radiological progression in RA.

Functional and prognostic significance of the genomic amplification of frizzled 6 (FZD6) in breast cancer

Frizzled receptors mediate Wnt ligand signalling, which is crucially involved in regulating tissue development and differentiation, and is often deregulated in cancer. In this study, we found that the gene encoding the Wnt receptor frizzled 6 (FZD6) is frequently amplified in breast cancer, with an increased incidence in the triple‐negative breast cancer (TNBC) subtype. Ablation of FZD6 expression in mammary cancer cell lines: (1) inhibited motility and invasion; (2) induced a more symmetrical shape of organoid three‐dimensional cultures; and (3) inhibited bone and liver metastasis in vivo. Mechanistically, FZD6 signalling is required for the assembly of the fibronectin matrix, interfering with the organization of the actin cytoskeleton. Ectopic delivery of fibronectin in FZD6‐depleted, triple‐negative MDA‐MB‐231 cells rearranged the actin cytoskeleton and restored epidermal growth factor‐mediated invasion. In patients with localized, lymph node‐negative (early) breast cancer, positivity of tumour cells for FZD6 protein identified patients with reduced distant relapse‐free survival. Multivariate analysis indicated an independent prognostic significance of FZD6 expression in TNBC tumours, predicting distant, but not local, relapse. We conclude that the FZD6–fibronectin actin axis identified in our study could be exploited for drug development in highly metastatic forms of breast cancer, such as TNBC.

Functional redundancy between Apc and Apc2 regulates tissue homeostasis and prevents tumorigenesis in murine mammary epithelium

Aberrant Wnt signaling within breast cancer is associated with poor prognosis, but regulation of this pathway in breast tissue remains poorly understood and the consequences of immediate or long-term dysregulation remain elusive. The exact contribution of the Wnt-regulating proteins adenomatous polyposis coli (APC) and APC2 in the pathogenesis of human breast cancer are ill-defined, but our analysis of publically available array data sets indicates that tumors with concomitant low expression of both proteins occurs more frequently in the ‘triple negative’ phenotype, which is a subtype of breast cancer with particularly poor prognosis. We have used mouse transgenics to delete Apc and/or Apc2 from mouse mammary epithelium to elucidate the significance of these proteins in mammary homeostasis and delineate their influences on Wnt signaling and tumorigenesis. Loss of either protein alone failed to affect Wnt signaling levels or tissue homeostasis. Strikingly, concomitant loss led to local disruption of β-catenin status, disruption in epithelial integrity, cohesion and polarity, increased cell division and a distinctive form of ductal hyperplasia with ‘squamoid’ ghost cell nodules in young animals. Upon aging, the development of Wnt activated mammary carcinomas with squamous differentiation was accompanied by a significantly reduced survival. This novel Wnt-driven mammary tumor model highlights the importance of functional redundancies existing between the Apc proteins both in normal homeostasis and in tumorigenesis.

A four-gene decision tree signature classification of triple-negative breast cancer: Implications for targeted therapeutics

The molecular complexity of triple-negative breast cancers (TNBCs) provides a challenge for patient management. We set out to characterize this heterogeneous disease by combining transcriptomics and genomics data, with the aim of revealing convergent pathway dependencies with the potential for treatment intervention. A Bayesian algorithm was used to integrate molecular profiles in two TNBC cohorts, followed by validation using five independent cohorts (n = 1,168), including three clinical trials. A four-gene decision tree signature was identified, which robustly classified TNBCs into six subtypes. All four genes in the signature (EXO1, TP53BP2, FOXM1, and RSU1) are associated with either genomic instability, malignant growth, or treatment response. One of the six subtypes, MC6, encompassed the largest proportion of tumors (∼50%) in early diagnosed TNBCs. In TNBC patients with metastatic disease, the MC6 proportion was reduced to 25%, and was independently associated with a higher response rate to platinum-based chemotherapy. In TNBC cell line data, platinum sensitivity was recapitulated, and a sensitivity to the inhibition of the phosphatase PPM1D was revealed. Molecularly, MC6-TNBCs displayed high levels of telomeric allelic imbalances, enrichment of CD4+ and CD8+ immune signatures, and reduced expression of genes negatively regulating the MAPK signaling pathway. These observations suggest that our integrative classification approach may identify TNBC patients with discernible and theoretically pharmacologically tractable features that merit further studies in prospective trials.

Abstract P1-07-03: Mesenchymal subtype negatively associates with the presence of immune infiltrates within a triple negative breast cancer classifier

Introduction: Lehmann and colleagues (Lehmann et al., 2011) devised a gene expression classification system for triple negative breast cancer (TNBC) consisting of seven subtypes—IM, BL1, BL2, LAR, M, MSL, and UNS (unselected). We (Ring et al., 2016) recently modified this original algorithm of 2188 gene subtyping into a 101-gene algorithm. In addition to a reduction of genes, the 101-gene algorithm has two methodological differences: first, the immunomodulatory (IM) signature was treated not as a subtype but rather as a binary feature of one of the other subtypes (e.g. BL1/IM+, LAR/IM-); second, when tumors—by a predefined correlation coefficient—showed traits of more than one subtype, both subtypes were reported as “dual subtypes.” Aim: Our aim was to apply the 101-gene algorithm for TNBC subtyping and to establish the relation of TNBC subtypes with their IM-status across several independent data sets. Methods: 951 patients from four independent TNBC cohorts with available gene expression data were analyzed by the 101-gene algorithm. Of these 848 were classified with at least one subtype. Results: The distribution of the 5 TNBC subtypes in both single and dual subtypes was 47%,10%,15%,18%,11%, for BL1, BL2, LAR, M, and MSL respectively. The majority of cases gave only one subtype (572, 67%) with M (Mesenchymal) being 9% (n=54) of these. Given this frequency of 9% of M as a baseline, in the remaining 276 (33%) cases with dual subtypes, the expectation that M would be one of the two is 11% (64 subtype calls). However, M is one of the two of the dual subtypes at a much higher frequency of 40% (222 subtype calls, Chi-Squared, P Conclusions: We further have confirmed with the 101-gene algorithm that the IM signature inversely associates with the M subtype as it has been observed with the 2188 gene algorithm (Lehmann et al., 2016). Moreover, the M signature is occasionally a confounder of other subtypes however still identifies those tumors negative for immune infiltrates. This raises important opportunities to understand the relationships between intrinsic tumor biology reflected in TNBC subtypes and their interaction with variable immune cell stroma which are the subject of ongoing analyses. Citation Format: Grigoriadis A, Quist J, Mirza H, Cheang MC, Ring BZ, Hout DR, Bailey DB, Seitz RS, Tutt AN. Mesenchymal subtype negatively associates with the presence of immune infiltrates within a triple negative breast cancer classifier [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-03.

Functional redundancy between Apc and Apc2 regulates tissue homeostasis and prevents tumourigenesis in murine mammary epithelium

Aberrant Wnt signaling within breast cancer is associated with poor prognosis, but regulation of this pathway in breast tissue remains poorly understood and the consequences of immediate or long-term dysregulation remain elusive. The exact contribution of the Wnt-regulating proteins adenomatous polyposis coli (APC) and APC2 in the pathogenesis of human breast cancer are ill-defined, but our analysis of publically available array data sets indicates that tumors with concomitant low expression of both proteins occurs more frequently in the ‘triple negative’ phenotype, which is a subtype of breast cancer with particularly poor prognosis. We have used mouse transgenics to delete Apc and/or Apc2 from mouse mammary epithelium to elucidate the significance of these proteins in mammary homeostasis and delineate their influences on Wnt signaling and tumorigenesis. Loss of either protein alone failed to affect Wnt signaling levels or tissue homeostasis. Strikingly, concomitant loss led to local disruption of β-catenin status, disruption in epithelial integrity, cohesion and polarity, increased cell division and a distinctive form of ductal hyperplasia with ‘squamoid’ ghost cell nodules in young animals. Upon aging, the development of Wnt activated mammary carcinomas with squamous differentiation was accompanied by a significantly reduced survival. This novel Wnt-driven mammary tumor model highlights the importance of functional redundancies existing between the Apc proteins both in normal homeostasis and in tumorigenesis.

Abstract 178: PIM1, a novel target in chemotherapy-resistant triple-negative breast cancer

Triple-Negative Breast Cancers (TNBCs) are aggressive, associated with poor prognosis and lack targeted therapies, mainly due to the absence of suitable molecular targets. The genomic region 6p21-p25 is recurrently amplified in TNBCs and encompasses the PIM1 oncogene. This study interrogated genomic breast cancer datasets and identified gene copy number-driven increase of PIM1 expression in TNBC. To understand the role of PIM1 in malignant phenotypes of TNBCs, functional studies were carried out in breast cancer and non-malignant mammary epithelial cell line models. RNA interference and rescue-of-function experiments revealed addiction to PIM1 kinase for cell population growth and apoptosis protection in TNBC cells, absent in non-malignant cells. In cells sensitive to PIM1 knockdown, we observed a subsequent reduction of the expression of the anti-apoptotic proteins BCL2 and MCL1. Moreover, exogenous expression of BCL2 prevented apoptosis caused by PIM1 knockdown. BH3-profiling analysis further confirmed that PIM1 blocks apoptosis elicited through the mitochondrial pathway in TNBC cell lines. The activity of PIM1 in other cancers has been closely linked to the regulation of c-MYC, an “un-targetable” oncogene that drives malignancy and that is frequently amplified and highly expressed in primary TNBCs and post-chemotherapy residual disease. Importantly, we found that PIM1 expression associates with several MYC-transcriptional signatures in TNBCs and mediates phosphorylation of c-MYC Ser62 and Histone-H3 Ser10, key targets for MYC-driven transcription. Exogenous expression of MYC rescued the phenotypes caused by PIM1 knockdown, providing mechanistic insights for the observed addiction. Finally, the therapeutic potential of targeting PIM1 was assessed using AZD1208, a small molecule inhibitor of the PIM kinase family. AZD1208 inhibited growth and sensitized TNBC derived cell lines, cell line xenografts and PDXs to chemotherapy by increasing apoptosis. We therefore identify PIM1 as a driver of malignancy in TNBC, illustrating relationships with MYC-activation and regulation of anti-apoptotic BCL2 and MCL1 proteins. PIM1 inhibitors could provide a potential therapeutic to abrogate chemotherapy resistance in TNBCs. Citation Format: Fara Braso-Maristany, Simone Filosto, Steven Catchpole, Rebecca Marlow, Jelmar Quist, Erika Francesch Domenech, Gianmaria Liccardi, Leila Zakka, Violeta Serra, Albert Gris, Maggie Cheang, Nirmesh Patel, Anna Perdrix Rosell, Patrycja Gazinska, Elodie Noel, Jonathan Watkins, Pierfrancesco Marra, Anita Grigoriadis, Andrew Tutt. PIM1, a novel target in chemotherapy-resistant triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 178.

Abstract 67: An integrated copy number and gene expression genomics analysis and RNAi approach identifies and validates the KIFC1 kinesin as a malignant cell selective target in triple negative breast cancer

Triple negative breast cancers (TNBCs) lack oestrogen (ER), progesterone (PR) and human epidermal growth factor 2 (HER2) receptors, and have limited targeted treatment options. Large scale genomic and transcriptomic studies have advanced our understanding of the changes which occur in TNBCs. However, the substantial number of copy number and gene expression alterations present in TNBCs makes it difficult to identify putative drivers, biomarkers and/or therapeutic targets of the disease. To overcome this problem, we have carried out an integrative computational and RNAi based approach to identify genes required for proliferation of TNBC. Copy number and gene expression alterations were analysed using Affymetrix Human Exon HTS1.0 and SNP6.0 data of 152 primary breast tumours enriched for a TNBC phenotype and 9 normal breast epithelium. These analyses revealed 141 candidate genes whose upregulated gene expression is copy number driven in TNBC. The functional dependence on each of these genes was subsequently examined using RNAi in an array of 17 breast cancer and non-malignant cell lines using 6-8 cell lines per gene covering the widest possible range of expression levels for that gene. We validated a malignant cell specific functional dependence on 37 of the 141 genes using this method. STRING analysis of validated hits reveals a subset of genes involved in the process of cell division and mitosis including the previously characterised mitotic kinase TTK. Of these, we further validated KIFC1 (HSET) which is known to play a role in clustering supernumerary centrosomes, a common occurrence in breast cancer. We show that siRNA and shRNA mediated depletion of KIFC1 decreases cell viability and clonogenic ability specifically in centrosome amplified cell lines, which can be rescued upon introduction of an si/shRNA resistant KIFC1. KIFC1 depletion also produces a high level of catastrophic multipolar mitoses in centrosome amplified but not in non-amplified cell lines. Furthermore, in-vivo studies show that inducible depletion of KIFC1 suppresses tumour growth of centrosome amplified cell line xenografts. Our work has identified and functionally validated novel drivers, and potential therapeutic targets in TNBC. The data presented here shows KIFC1, a druggable kinesin motor protein is a promising target for therapeutic intervention being expressed through gene copy gain in a significant proportion of TNBCs. We validate its role in cancer-specific amplified centrosome clustering showing KIFC1 plays an essential role in aiding the survival of breast cancer cells that have supernumerary centrosomes in both in-vitro and in-vivo contexts. Citation Format: Nirmesh S. Patel, Konstantinos Drosopoulos, Daniel Weekes, Elodie Noel, Hasan Mirza, Mamunur Rashid, Emanuele de Rinaldis, Fara Braso Maristany, Sumi Mathew, Erika Francesch Domenech, Patrycja Gazinska, Farzana Noor, Jelmar Quist, Rebecca Marlow, Anita Grigoriadis, Spiros Linardopoulos, Andrew N. Tutt. An integrated copy number and gene expression genomics analysis and RNAi approach identifies and validates the KIFC1 kinesin as a malignant cell selective target in triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 67.