MDM2 inhibition in combination with endocrine therapy and CDK4/6 inhibition for the treatment of ER-positive breast cancer.

BACKGROUND: Resistance to endocrine therapy is a major clinical challenge in the management of oestrogen receptor (ER)-positive breast cancer. In this setting, p53 is frequently wildtype and its activity may be suppressed via upregulation of its key regulator MDM2. This underlies our rationale to evaluate MDM2 inhibition as a therapeutic strategy in treatment-resistant ER-positive breast cancer. METHODS: We used the MDM2 inhibitor NVP-CGM097 to treat in vitro and in vivo models alone and in combination with fulvestrant or palbociclib. We perform cell viability, cell cycle, apoptosis and senescence assays to evaluate anti-tumour effects in p53 wildtype and p53 mutant ER-positive cell lines (MCF-7, ZR75-1, T-47D) and MCF-7 lines resistant to endocrine therapy and to CDK4/6 inhibition. We further assess the drug effects in patient-derived xenograft (PDX) models of endocrine-sensitive and endocrine-resistant ER-positive breast cancer. RESULTS: We demonstrate that MDM2 inhibition results in cell cycle arrest and increased apoptosis in p53-wildtype in vitro and in vivo breast cancer models, leading to potent anti-tumour activity. We find that endocrine therapy or CDK4/6 inhibition synergises with MDM2 inhibition but does not further enhance apoptosis. Instead, combination treatments result in profound regulation of cell cycle-related transcriptional programmes, with synergy achieved through increased antagonism of cell cycle progression. Combination therapy pushes cell lines resistant to fulvestrant or palbociclib to become senescent and significantly reduces tumour growth in a fulvestrant-resistant patient-derived xenograft model. CONCLUSIONS: We conclude that MDM2 inhibitors in combination with ER degraders or CDK4/6 inhibitors represent a rational strategy for treating advanced, endocrine-resistant ER-positive breast cancer, operating through synergistic activation of cell cycle co-regulatory programmes.

Protein diversity in discrete structures at the distal tip of the trypanosome flagellum.

The distal end of the eukaryotic flagellum/cilium is important for axonemal growth and signaling and has distinct biomechanical properties. Specific flagellum tip structures exist, yet their composition, dynamics, and functions are largely unknown. We used biochemical approaches to identify seven constituents of the flagella connector at the tip of an assembling trypanosome flagellum and three constituents of the axonemal capping structure at the tips of both assembling and mature flagella. Both tip structures contain evolutionarily conserved as well as kinetoplastid-specific proteins, and component assembly into the structures occurs very early during flagellum extension. Localization and functional studies reveal that the flagella connector membrane junction is attached to the tips of extending microtubules of the assembling flagellum by a kinesin-15 family member. On the opposite side, a kinetoplastid-specific kinesin facilitates attachment of the junction to the microtubules in the mature flagellum. Functional studies also suggest roles of several other components and the definition of subdomains in the tip structures.

Targeting promiscuous heterodimerization overcomes innate resistance to ERBB2 dimerization inhibitors in breast cancer.

BACKGROUND: The oncogenic receptor tyrosine kinase (RTK) ERBB2 is known to dimerize with other EGFR family members, particularly ERBB3, through which it potently activates PI3K signalling. Antibody-mediated inhibition of this ERBB2/ERBB3/PI3K axis has been a cornerstone of treatment for ERBB2-amplified breast cancer patients for two decades. However, the lack of response and the rapid onset of relapse in many patients now question the assumption that the ERBB2/ERBB3 heterodimer is the sole relevant effector target of these therapies. METHODS: Through a systematic protein-protein interaction screen, we have identified and validated alternative RTKs that interact with ERBB2. Using quantitative readouts of signalling pathway activation and cell proliferation, we have examined their influence upon the mechanism of trastuzumab- and pertuzumab-mediated inhibition of cell growth in ERBB2-amplified breast cancer cell lines and a patient-derived xenograft model. RESULTS: We now demonstrate that inactivation of ERBB3/PI3K by these therapeutic antibodies is insufficient to inhibit the growth of ERBB2-amplified breast cancer cells. Instead, we show extensive promiscuity between ERBB2 and an array of RTKs from outside of the EGFR family. Paradoxically, pertuzumab also acts as an artificial ligand to promote ERBB2 activation and ERK signalling, through allosteric activation by a subset of these non-canonical RTKs. However, this unexpected activation mechanism also increases the sensitivity of the receptor network to the ERBB2 kinase inhibitor lapatinib, which in combination with pertuzumab, displays a synergistic effect in single-agent resistant cell lines and PDX models. CONCLUSIONS: The interaction of ERBB2 with a number of non-canonical RTKs activates a compensatory signalling response following treatment with pertuzumab, although a counter-intuitive combination of ERBB2 antibody therapy and a kinase inhibitor can overcome this innate therapeutic resistance.

A Role for Chlorinated Nucleosides in the Perturbation of Macrophage Function and Promotion of Inflammation.

During inflammation, myeloperoxidase released from activated phagocytes generates the highly reactive oxidant hypochlorous acid (HOCl). This oxidant plays an important role in the immune response but can also promote tissue damage and has been strongly linked with the development of numerous inflammatory diseases. HOCl reacts with cellular DNA forming chlorinated nucleobases, which induce strand breaks, mutations, and cross-links. Although it has been shown that chlorinated nucleosides are present within inflammatory pathologies and diseased tissue, whether or not these species are biomarkers formed as a byproduct of chronic inflammation or play a role in the disease progression has not been ascertained. In this study, we show that exposure of J774A.1 macrophage-like cells to chlorinated ribose and deoxyribose nucleosides results in the incorporation of 5-chloro-cytidine (5ClC), 8-chloro-adenosine (8ClA), and 8-chloro-guanosine (8ClG) into the cellular RNA and 5-chloro-deoxycytidine (5CldC) but not 8-chloro-deoxyguanosine (8CldG) or 8-chloro-deoxyadenosine (8CldA) into cellular DNA. Evidence was obtained for the clearance of 5ClC from the RNA, with a loss of 8ClA and 8ClG observed to a lesser extent, whereas an increase in the level of 5CldC in DNA was seen on further incubation of treated cells in the absence of chlorinated nucleosides. Importantly, exposure of the macrophages to chlorinated nucleosides, particularly 8ClG and 5ClC, resulted in the increased expression of interleukin-1ß, and other pro-inflammatory cytokines and chemokines. With 5ClC, this inflammatory response was associated with the increased nuclear translocation of the NF-κB subunit, p65, rather than inflammasome activation. This alteration in gene expression appeared to be unrelated to the extent of incorporation of the chlorinated nucleosides into RNA or DNA and was not associated with any significant changes in cell viability or proliferation. Taken together, these results highlight a potential biological role for chlorinated nucleosides to promote inflammatory disease, in addition to their utility as biomarkers.

Evidence of intraflagellar transport and apical complex formation in a free-living relative of the apicomplexa.

Since its first description, Chromera velia has attracted keen interest as the closest free-living relative of parasitic Apicomplexa. The life cycle of this unicellular alga is complex and involves a motile biflagellate form. Flagella are thought to be formed in the cytoplasm, a rare phenomenon shared with Plasmodium in which the canonical mode of flagellar assembly, intraflagellar transport, is dispensed with. Here we demonstrate the expression of intraflagellar transport components in C. velia, answering the question of whether this organism has the potential to assemble flagella via the canonical route. We have developed and characterized a culturing protocol that favors the generation of flagellate forms. From this, we have determined a marked shift in the mode of daughter cell production from two to four daughter cells per division as a function of time after passage. We conduct an ultrastructural examination of the C. velia flagellate form by using serial TEM and show that flagellar biogenesis in C. velia occurs prior to cytokinesis. We demonstrate a close association of the flagellar apparatus with a complex system of apical structures, including a micropore, a conoid, and a complex endomembrane system reminiscent of the apical complex of parasitic apicomplexans. Recent work has begun to elucidate the possible flagellar origins of the apical complex, and we show that in C. velia these structures are contemporaneous within a single cell and share multiple connections. We propose that C. velia therefore represents a vital piece in the puzzle of the origins of the apical complex.

The potential of epigenetic therapy to target the 3D epigenome in endocrine-resistant breast cancer.

Three-dimensional (3D) epigenome remodeling is an important mechanism of gene deregulation in cancer. However, its potential as a target to counteract therapy resistance remains largely unaddressed. Here, we show that epigenetic therapy with decitabine (5-Aza-mC) suppresses tumor growth in xenograft models of pre-clinical metastatic estrogen receptor positive (ER+) breast tumor. Decitabine-induced genome-wide DNA hypomethylation results in large-scale 3D epigenome deregulation, including de-compaction of higher-order chromatin structure and loss of boundary insulation of topologically associated domains. Significant DNA hypomethylation associates with ectopic activation of ER-enhancers, gain in ER binding, creation of new 3D enhancer-promoter interactions and concordant up-regulation of ER-mediated transcription pathways. Importantly, long-term withdrawal of epigenetic therapy partially restores methylation at ER-enhancer elements, resulting in a loss of ectopic 3D enhancer-promoter interactions and associated gene repression. Our study illustrates the potential of epigenetic therapy to target ER+ endocrine-resistant breast cancer by DNA methylation-dependent rewiring of 3D chromatin interactions, which are associated with the suppression of tumor growth.

Proteomics and the Trypanosoma brucei cytoskeleton: advances and opportunities.

Trypanosoma brucei is the etiological agent of devastating parasitic disease in humans and livestock in sub-saharan Africa. The pathogenicity and growth of the parasite are intimately linked to its shape and form. This is in turn derived from a highly ordered microtubule cytoskeleton that forms a tightly arrayed cage directly beneath the pellicular membrane and numerous other cytoskeletal structures such as the flagellum. The parasite undergoes extreme changes in cellular morphology during its life cycle and cell cycles which require a high level of integration and coordination of cytoskeletal processes. In this review we will discuss the role that proteomics techniques have had in advancing our understanding of the molecular composition of the cytoskeleton and its functions. We then consider future opportunities for the application of these techniques in terms of addressing some of the unanswered questions of trypanosome cytoskeletal cell biology with particular focus on the differences in the composition and organisation of the cytoskeleton through the trypanosome life-cycle.

Flagellar motility is required for the viability of the bloodstream trypanosome.

The 9 + 2 microtubule axoneme of flagella and cilia represents one of the most iconic structures built by eukaryotic cells and organisms. Both unity and diversity are present among cilia and flagella on the evolutionary as well as the developmental scale. Some cilia are motile, whereas others function as sensory organelles and can variously possess 9 + 2 and 9 + 0 axonemes and other associated structures. How such unity and diversity are reflected in molecular repertoires is unclear. The flagellated protozoan parasite Trypanosoma brucei is endemic in sub-Saharan Africa, causing devastating disease in humans and other animals. There is little hope of a vaccine for African sleeping sickness and a desperate need for modern drug therapies. Here we present a detailed proteomic analysis of the trypanosome flagellum. RNA interference (RNAi)-based interrogation of this proteome provides functional insights into human ciliary diseases and establishes that flagellar function is essential to the bloodstream-form trypanosome. We show that RNAi-mediated ablation of various proteins identified in the trypanosome flagellar proteome leads to a rapid and marked failure of cytokinesis in bloodstream-form (but not procyclic insect-form) trypanosomes, suggesting that impairment of flagellar function may provide a method of disease control. A postgenomic meta-analysis, comparing the evolutionarily ancient trypanosome with other eukaryotes including humans, identifies numerous trypanosome-specific flagellar proteins, suggesting new avenues for selective intervention.

Epigenetic Therapies and Biomarkers in Breast Cancer.

Epigenetic therapies remain a promising, but still not widely used, approach in the management of patients with cancer. To date, the efficacy and use of epigenetic therapies has been demonstrated primarily in the management of haematological malignancies, with limited supportive data in solid malignancies. The most studied epigenetic therapies in breast cancer are those that target DNA methylation and histone modification; however, none have been approved for routine clinical use. The majority of pre-clinical and clinical studies have focused on triple negative breast cancer (TNBC) and hormone-receptor positive breast cancer. Even though the use of epigenetic therapies alone in the treatment of breast cancer has not shown significant clinical benefit, these therapies show most promise in use in combinations with other treatments. With improving technologies available to study the epigenetic landscape in cancer, novel epigenetic alterations are increasingly being identified as potential biomarkers of response to conventional and epigenetic therapies. In this review, we describe epigenetic targets and potential epigenetic biomarkers in breast cancer, with a focus on clinical trials of epigenetic therapies. We describe alterations to the epigenetic landscape in breast cancer and in treatment resistance, highlighting mechanisms and potential targets for epigenetic therapies. We provide an updated review on epigenetic therapies in the pre-clinical and clinical setting in breast cancer, with a focus on potential real-world applications. Finally, we report on the potential value of epigenetic biomarkers in diagnosis, prognosis and prediction of response to therapy, to guide and inform the clinical management of breast cancer patients.

MDM2 as a Rational Target for Intervention in CDK4/6 Inhibitor Resistant, Hormone Receptor Positive Breast Cancer.

With the adoption of inhibitors of cyclin dependent kinases 4 and 6 (CDK4/6i) in combination with endocrine therapy as standard of care for the treatment of advanced and metastatic estrogen receptor positive (ER+) breast cancer, the search is now on for novel therapeutic options to manage the disease after the inevitable development of resistance to CDK4/6i. In this review we will consider the integral role that the p53/MDM2 axis plays in the interactions between CDK4/6, ERα, and inhibitors of these molecules, the current preclinical evidence for the efficacy of MDM2 inhibitors in ER+ breast cancer, and discuss the possibility of targeting the p53/MDM2 via inhibition of MDM2 in the CDK4/6i resistance setting.

Molecular Biomarkers for Contemporary Therapies in Hormone Receptor-Positive Breast Cancer.

Systemic treatment of hormone receptor-positive (HR+) breast cancer is undergoing a renaissance, with a number of targeted therapies including CDK4/6, mTOR, and PI3K inhibitors now approved for use in combination with endocrine therapies. The increased use of targeted therapies has changed the natural history of HR+ breast cancers, with the emergence of new escape mechanisms leading to the inevitable progression of disease in patients with advanced cancers. The identification of new predictive and pharmacodynamic biomarkers to current standard-of-care therapies and discovery of new therapies is an evolving and urgent clinical challenge in this setting. While traditional, routinely measured biomarkers such as estrogen receptors (ERs), progesterone receptors (PRs), and human epidermal growth factor receptor 2 (HER2) still represent the best prognostic and predictive biomarkers for HR+ breast cancer, a significant proportion of patients either do not respond to endocrine therapy or develop endocrine resistant disease. Genomic tests have emerged as a useful adjunct prognostication tool and guide the addition of chemotherapy to endocrine therapy. In the treatment-resistant setting, mutational profiling has been used to identify ESR1, PIK3CA, and AKT mutations as predictive molecular biomarkers to newer therapies. Additionally, pharmacodynamic biomarkers are being increasingly used and considered in the metastatic setting. In this review, we summarise the current state-of-the-art therapies; prognostic, predictive, and pharmacodynamic molecular biomarkers; and how these are impacted by emerging therapies for HR+ breast cancer.

Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer.

CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy have shown impressive efficacy in estrogen receptor-positive advanced breast cancer. However, most patients will eventually experience disease progression on this combination, underscoring the need for effective subsequent treatments or better initial therapies. Here, we show that triple inhibition with fulvestrant, CDK4/6i and AKT inhibitor (AKTi) durably impairs growth of breast cancer cells, prevents progression and reduces metastasis of tumor xenografts resistant to CDK4/6i-fulvestrant combination or fulvestrant alone. Importantly, switching from combined fulvestrant and CDK4/6i upon resistance to dual combination with AKTi and fulvestrant does not prevent tumor progression. Furthermore, triple combination with AKTi significantly inhibits growth of patient-derived xenografts resistant to combined CDK4/6i and fulvestrant. Finally, high phospho-AKT levels in metastasis of breast cancer patients treated with a combination of CDK4/6i and endocrine therapy correlates with shorter progression-free survival. Our findings support the clinical development of ER, CDK4/6 and AKT co-targeting strategies following progression on CDK4/6i and endocrine therapy combination, and in tumors exhibiting high phospho-AKT levels, which are associated with worse clinical outcome.

Synergistic targeting of BRCA1 mutated breast cancers with PARP and CDK2 inhibition.

Basal-like breast cancers (BLBC) are aggressive breast cancers that respond poorly to targeted therapies and chemotherapies. In order to define therapeutically targetable subsets of BLBC we examined two markers: cyclin E1 and BRCA1 loss. In high grade serous ovarian cancer (HGSOC) these markers are mutually exclusive, and define therapeutic subsets. We tested the same hypothesis for BLBC. Using a BLBC cohort enriched for BRCA1 loss, we identified convergence between BRCA1 loss and high cyclin E1 protein expression, in contrast to HGSOC in which CCNE1 amplification drives increased cyclin E1. In cell lines, BRCA1 loss was associated with stabilized cyclin E1 during the cell cycle, and BRCA1 siRNA led to increased cyclin E1 in association with reduced phospho-cyclin E1 T62. Mutation of cyclin E1 T62 to alanine increased cyclin E1 stability. We showed that tumors with high cyclin E1/BRCA1 mutation in the BLBC cohort also had decreased phospho-T62, supporting this hypothesis. Since cyclin E1/CDK2 protects cells from DNA damage and cyclin E1 is elevated in BRCA1 mutant cancers, we hypothesized that CDK2 inhibition would sensitize these cancers to PARP inhibition. CDK2 inhibition induced DNA damage and synergized with PARP inhibitors to reduce cell viability in cell lines with homologous recombination deficiency, including BRCA1 mutated cell lines. Treatment of BRCA1 mutant BLBC patient-derived xenograft models with combination PARP and CDK2 inhibition led to tumor regression and increased survival. We conclude that BRCA1 status and high cyclin E1 have potential as predictive biomarkers to dictate the therapeutic use of combination CDK inhibitors/PARP inhibitors in BLBC.

DNA methylation is required to maintain both DNA replication timing precision and 3D genome organization integrity.

DNA replication timing and three-dimensional (3D) genome organization are associated with distinct epigenome patterns across large domains. However, whether alterations in the epigenome, in particular cancer-related DNA hypomethylation, affects higher-order levels of genome architecture is still unclear. Here, using Repli-Seq, single-cell Repli-Seq, and Hi-C, we show that genome-wide methylation loss is associated with both concordant loss of replication timing precision and deregulation of 3D genome organization. Notably, we find distinct disruption in 3D genome compartmentalization, striking gains in cell-to-cell replication timing heterogeneity and loss of allelic replication timing in cancer hypomethylation models, potentially through the gene deregulation of DNA replication and genome organization pathways. Finally, we identify ectopic H3K4me3-H3K9me3 domains from across large hypomethylated domains, where late replication is maintained, which we purport serves to protect against catastrophic genome reorganization and aberrant gene transcription. Our results highlight a potential role for the methylome in the maintenance of 3D genome regulation.

Targeting CDK2 in cancer: challenges and opportunities for therapy.

Cyclin-dependent kinase 2 (CDK2) plays a pivotal part in cell cycle regulation and is involved in a range of biological processes. CDK2 interacts with and phosphorylates proteins in pathways such as DNA damage, intracellular transport, protein degradation, signal transduction, DNA and RNA metabolism and translation. CDK2 and its regulatory subunits are deregulated in many human cancers and there is emerging evidence suggesting CDK2 inhibition elicits antitumor activity in a subset of tumors with defined genetic features. Previous CDK2 inhibitors were nonspecific and limited by off-target effects. The development of new-generation CDK2 inhibitors represents a therapeutic opportunity for CDK2-dependent cancers.

Analysis of the trypanosome flagellar proteome using a combined electron transfer/collisionally activated dissociation strategy.

The use of electron-transfer dissociation as an alternative peptide ion activation method for generation of protein sequence information is examined here in comparison with the conventional method of choice, collisionally activated dissociation, using a linear ion trapping instrument. Direct comparability between collisionally and electron-transfer-activated product ion data were ensured by employing an activation-switching method during acquisition, sequentially activating precisely the same precursor ion species with each fragmentation method in turn. Sequest (Thermo Fisher Scientific, San Jose, CA) searching of product ion data generated an overlapping yet distinct pool of polypeptide identifications from the products of collisional and electron-transfer-mediated activation products. To provide a highly confident set of protein recognitions, identification data were filtered using parameters that achieved a peptide false discovery rate of 1%, with two or more independent peptide assignments required for each protein. The use of electron transfer dissociation (ETD) has allowed us to identify additional peptides where the quality of product ion data generated by collisionally activated dissociation (CAD) was insufficient to infer peptide sequence. Thus, a combined ETD/CAD approach leads to the recognition of more peptides and proteins than are achieved using peptide analysis by CAD- or ETD-based tandem mass spectrometry alone.

Overcoming CDK4/6 inhibitor resistance in ER-positive breast cancer.

Three inhibitors of CDK4/6 kinases were recently FDA approved for use in combination with endocrine therapy, and they significantly increase the progression-free survival of patients with advanced estrogen receptor-positive (ER+) breast cancer in the first-line treatment setting. As the new standard of care in some countries, there is the clinical emergence of patients with breast cancer that is both CDK4/6 inhibitor and endocrine therapy resistant. The strategies to combat these cancers with resistance to multiple treatments are not yet defined and represent the next major clinical challenge in ER+ breast cancer. In this review, we discuss how the molecular landscape of endocrine therapy resistance may affect the response to CDK4/6 inhibitors, and how this intersects with biomarkers of intrinsic insensitivity. We identify the handful of pre-clinical models of acquired resistance to CDK4/6 inhibitors and discuss whether the molecular changes in these models are likely to be relevant or modified in the context of endocrine therapy resistance. Finally, we consider the crucial question of how some of these changes are potentially amenable to therapy.

Non-canonical AR activity facilitates endocrine resistance in breast cancer.

The role of androgen receptor (AR) in endocrine-resistant breast cancer is controversial and clinical trials targeting AR with an AR antagonist (e.g., enzalutamide) have been initiated. Here, we investigated the consequence of AR antagonism using in vitro and in vivo models of endocrine resistance. AR antagonism in MCF7-derived tamoxifen-resistant (TamR) and long-term estrogen-deprived breast cancer cell lines were achieved using siRNA-mediated knockdown or pharmacological inhibition with enzalutamide. The efficacy of enzalutamide was further assessed in vivo in an estrogen-independent endocrine-resistant patient-derived xenograft (PDX) model. Knockdown of AR inhibited the growth of the endocrine-resistant cell line models. Microarray gene expression profiling of the TamR cells following AR knockdown revealed perturbations in proliferative signaling pathways upregulated in endocrine resistance. AR loss also increased some canonical ER signaling events and restored sensitivity of TamR cells to tamoxifen. In contrast, enzalutamide did not recapitulate the effect of AR knockdown in vitro, even though it inhibited canonical AR signaling, which suggests that it is the non-canonical AR activity that facilitated endocrine resistance. Enzalutamide had demonstrable efficacy in inhibiting AR activity in vivo but did not affect the growth of the endocrine-resistant PDX model. Our findings implicate non-canonical AR activity in facilitating an endocrine-resistant phenotype in breast cancer. Unlike canonical AR signaling which is inhibited by enzalutamide, non-canonical AR activity is not effectively antagonized by enzalutamide, and this has important implications in the design of future AR-targeted clinical trials in endocrine-resistant breast cancer.