Targeting the Breast Cancer Kinome.

Protein kinases are highly tractable targets for the treatment of many cancers including breast cancer, due to their essential role in tumor cell proliferation and survival. Sequencing of the breast cancer genome and transcriptome has defined breast cancer as a heterogeneous disease that is classified into five molecular subtypes: luminal A, luminal B, HER2-enriched, basal-like, and claudin-low. Each subtype displays a unique expression profile of protein kinases that can be targeted by small molecule kinase inhibitors or biologics. An understanding of genomic changes, including mutations or copy number variations, for specific protein kinases and dependencies on kinases across breast cancer subtypes is allowing for a more rational design of targeted breast cancer therapies. While specific kinase inhibitors have had success in the clinic, including the CDK4/6 inhibitor palbociclib in combination with aromatase inhibitors in luminal breast cancer, patients often become resistant to treatment. An understanding of the mechanisms allowing cells to bypass targeted kinase inhibition has led to the development of combination therapies that are more durable in pre-clinical studies. However, the heterogeneity of resistance mechanisms and rapid adaptability of the kinome through feedback regulation greatly inhibit the long-term efficacy of combination kinase inhibitor therapies. It is becoming apparent that epigenetic inhibitors, such as HDAC and BET bromodomain inhibitors can block the transcriptional adaptability of tumor cells to kinase inhibitors and prevent the onset of resistance. Such novel combination therapies are currently showing promise in preclinical studies to markedly increase the durability of kinase inhibitors in breast cancer. J. Cell. Physiol. 232: 53-60, 2017. © 2016 Wiley Periodicals, Inc.

Crystal structures of a group II chaperonin reveal the open and closed states associated with the protein folding cycle.

Chaperonins are large protein complexes consisting of two stacked multisubunit rings, which open and close in an ATP-dependent manner to create a protected environment for protein folding. Here, we describe the first crystal structure of a group II chaperonin in an open conformation. We have obtained structures of the archaeal chaperonin from Methanococcus maripaludis in both a peptide acceptor (open) state and a protein folding (closed) state. In contrast with group I chaperonins, in which the equatorial domains share a similar conformation between the open and closed states and the largest motions occurs at the intermediate and apical domains, the three domains of the archaeal chaperonin subunit reorient as a single rigid body. The large rotation observed from the open state to the closed state results in a 65% decrease of the folding chamber volume and creates a highly hydrophilic surface inside the cage. These results suggest a completely distinct closing mechanism in the group II chaperonins as compared with the group I chaperonins.

Inhibition of unfolding and aggregation of lens protein human gamma D crystallin by sodium citrate.

Cataract affects 1 in 6 Americans over the age of 40, and represents a global health problem. Mature onset cataract is associated with the aggregation of partially unfolded or damaged proteins in the lens, which accumulate as an individual ages. Currently, surgery is the primary effective treatment for cataract. As an alternative preventive approach, small molecules have been suggested as potential therapeutic agents. In this work, we study the effect of sodium citrate on the stability of Human γD Crystallin (HγD-Crys), a structural protein of the eye lens, and two cataract-related mutants, L5S HγD-Crys and I90F HγD-Crys. In equilibrium unfolding-refolding studies, the presence of 250 mM sodium citrate increased the transition midpoint of the N-terminal domain (N-td) of WT HγD-Crys and L5S HγD-Crys by 0.3 M GuHCl, the C-terminal domain (C-td) by 0.6 M GuHCl, and the single transition of I90F HγD-Crys by 0.4 M GuHCl. In kinetic unfolding reactions, sodium citrate stabilization effect was observed only for the mutant I90F HγD-Crys. In the presence of citrate, a kinetic unfolding intermediate of I90F HγD-Crys was observed, which was not populated in the absence of citrate. The rates of aggregation were measured using solution turbidity. Sodium citrate demonstrated negligible effect on rate of aggregation of WT HγD-Crys, but considerably slowed the rate of aggregation of both L5S HγD-Crys and I90F HγD-Crys. The presence of sodium citrate dramatically slowed refolding of WT HγD-Crys and I90F HγD-Crys, but had a significantly smaller effect on the refolding of L5S HγD-Crys. The differential stabilizing effect of sodium citrate suggests that the ion is binding to a partially unfolded conformation of the C-td, but a solution-based Hofmeister effect cannot be eliminated as a possible explanation for the effects observed. These results indicate that assessment of potential anti-cataract agents needs to include effects on the unfolding and aggregation pathways, as well as the native state.

Microenvironmental IL-6 inhibits anti-cancer immune responses generated by cytotoxic chemotherapy.

Cytotoxic chemotherapeutics primarily function through DNA damage-induced tumor cell apoptosis, although the inflammation provoked by these agents can stimulate anti-cancer immune responses. The mechanisms that control these distinct effects and limit immunogenic responses to DNA-damage mediated cell death in vivo are currently unclear. Using a mouse model of BCR-ABL+ B-cell acute lymphoblastic leukemia, we show that chemotherapy-induced anti-cancer immunity is suppressed by the tumor microenvironment through production of the cytokine IL-6. The chemotherapeutic doxorubicin is curative in IL-6-deficient mice through the induction of CD8+ T-cell-mediated anti-cancer responses, while moderately extending lifespan in wild type tumor-bearing mice. We also show that IL-6 suppresses the effectiveness of immune-checkpoint inhibition with anti-PD-L1 blockade. Our results suggest that IL-6 is a key regulator of anti-cancer immune responses induced by genotoxic stress and that its inhibition can switch cancer cell clearance from primarily apoptotic to immunogenic, promoting and maintaining durable anti-tumor immune responses.

FOXA1 and adaptive response determinants to HER2 targeted therapy in TBCRC 036.

Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.

Discrete Adaptive Responses to MEK Inhibitor in Subpopulations of Triple-Negative Breast Cancer.

Triple-negative breast cancers contain a spectrum of epithelial and mesenchymal phenotypes. SUM-229PE cells represent a model for this heterogeneity, maintaining both epithelial and mesenchymal subpopulations that are genomically similar but distinct in gene expression profiles. We identified differential regions of open chromatin in epithelial and mesenchymal cells that were strongly correlated with regions of H3K27ac. Motif analysis of these regions identified consensus sequences for transcription factors that regulate cell identity. Treatment with the MEK inhibitor trametinib induced enhancer remodeling that is associated with transcriptional regulation of genes in epithelial and mesenchymal cells. Motif analysis of enhancer peaks downregulated in response to chronic treatment with trametinib identified AP-1 motif enrichment in both epithelial and mesenchymal subpopulations. Chromatin immunoprecipitation sequencing (ChIP-seq) of JUNB identified subpopulation-specific localization, which was significantly enriched at regions of open chromatin. These results indicate that cell identity controls localization of transcription factors and chromatin-modifying enzymes to enhancers for differential control of gene expression. We identified increased H3K27ac at an enhancer region proximal to CXCR7, a G-protein-coupled receptor that increased 15-fold in expression in the epithelial subpopulation during chronic treatment. RNAi knockdown of CXCR7 inhibited proliferation in trametinib-resistant cells. Thus, adaptive resistance to chronic trametinib treatment contributes to proliferation in the presence of the drug. Acquired amplification of KRAS following trametinib dose escalation further contributed to POS cell proliferation. Adaptive followed by acquired gene expression changes contributed to proliferation in trametinib-resistant cells, suggesting inhibition of early transcriptional reprogramming could prevent resistance and the bypass of targeted therapy. IMPLICATIONS: We defined the differential responses to trametinib in subpopulations of a clinically relevant in vitro model of TNBC, and identified both adaptive and acquired elements that contribute to the emergence of drug resistance mediated by increased expression of CXCR7 and amplification of KRAS.

Proteomic analysis defines kinase taxonomies specific for subtypes of breast cancer.

Multiplexed small molecule inhibitors covalently bound to Sepharose beads (MIBs) were used to capture functional kinases in luminal, HER2-enriched and triple negative (basal-like and claudin-low) breast cancer cell lines and tumors. Kinase MIB-binding profiles at baseline without perturbation proteomically distinguished the four breast cancer subtypes. Understudied kinases, whose disease associations and pharmacology are generally unexplored, were highly represented in MIB-binding taxonomies and are integrated into signaling subnetworks with kinases that have been previously well characterized in breast cancer. Computationally it was possible to define subtypes using profiles of less than 50 of the more than 300 kinases bound to MIBs that included understudied as well as metabolic and lipid kinases. Furthermore, analysis of MIB-binding profiles established potential functional annotations for these understudied kinases. Thus, comprehensive MIBs-based capture of kinases provides a unique proteomics-based method for integration of poorly characterized kinases of the understudied kinome into functional subnetworks in breast cancer cells and tumors that is not possible using genomic strategies. The MIB-binding profiles readily defined subtype-selective differential adaptive kinome reprogramming in response to targeted kinase inhibition, demonstrating how MIB profiles can be used in determining dynamic kinome changes that result in subtype selective phenotypic state changes.

Enhancer Remodeling during Adaptive Bypass to MEK Inhibition Is Attenuated by Pharmacologic Targeting of the P-TEFb Complex.

Targeting the dysregulated BRAF-MEK-ERK pathway in cancer has increasingly emerged in clinical trial design. Despite clinical responses in specific cancers using inhibitors targeting BRAF and MEK, resistance develops often involving nongenomic adaptive bypass mechanisms. Inhibition of MEK1/2 by trametinib in patients with triple-negative breast cancer (TNBC) induced dramatic transcriptional responses, including upregulation of receptor tyrosine kinases (RTK) comparing tumor samples before and after one week of treatment. In preclinical models, MEK inhibition induced genome-wide enhancer formation involving the seeding of BRD4, MED1, H3K27 acetylation, and p300 that drives transcriptional adaptation. Inhibition of the P-TEFb-associated proteins BRD4 and CBP/p300 arrested enhancer seeding and RTK upregulation. BRD4 bromodomain inhibitors overcame trametinib resistance, producing sustained growth inhibition in cells, xenografts, and syngeneic mouse TNBC models. Pharmacologic targeting of P-TEFb members in conjunction with MEK inhibition by trametinib is an effective strategy to durably inhibit epigenomic remodeling required for adaptive resistance.Significance: Widespread transcriptional adaptation to pharmacologic MEK inhibition was observed in TNBC patient tumors. In preclinical models, MEK inhibition induces dramatic genome-wide modulation of chromatin, in the form of de novo enhancer formation and enhancer remodeling. Pharmacologic targeting of P-TEFb complex members at enhancers is an effective strategy to durably inhibit such adaptation. Cancer Discov; 7(3); 302-21. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 235.

The group II chaperonin Mm-Cpn binds and refolds human γD crystallin.

Chaperonins assist in the folding of nascent and misfolded proteins, though the mechanism of folding within the lumen of the chaperonin remains poorly understood. The archeal chaperonin from Methanococcus marapaludis, Mm-Cpn, shares the eightfold double barrel structure with other group II chaperonins, including the eukaryotic TRiC/CCT, required for actin and tubulin folding. However, Mm-Cpn is composed of a single species subunit, similar to group I chaperonin GroEL, rather than the eight subunit species needed for TRiC/CCT. Features of the ß-sheet fold have been identified as sites of recognition by group II chaperonins. The crystallins, the major components of the vertebrate eye lens, are ß-sheet proteins with two homologous Greek key domains. During refolding in vitro a partially folded intermediate is populated, and partitions between productive folding and off-pathway aggregation. We report here that in the presence of physiological concentrations of ATP, Mm-Cpn suppressed the aggregation of HγD-Crys by binding the partially folded intermediate. The complex was sufficiently stable to permit recovery by size exclusion chromatography. In the presence of ATP, Mm-Cpn promoted the refolding of the HγD-Crys intermediates to the native state. The ability of Mm-Cpn to bind and refold a human ß-sheet protein suggests that Mm-Cpn may be useful as a simplified model for the substrate recognition mechanism of TRiC/CCT.