Metabolite Profiling Reveals the Glutathione Biosynthetic Pathway as a Therapeutic Target in Triple-Negative Breast Cancer.

Cancer cells can exhibit altered dependency on specific metabolic pathways and targeting these dependencies is a promising therapeutic strategy. Triple-negative breast cancer (TNBC) is an aggressive and genomically heterogeneous subset of breast cancer that is resistant to existing targeted therapies. To identify metabolic pathway dependencies in TNBC, we first conducted mass spectrometry-based metabolomics of TNBC and control cells. Relative levels of intracellular metabolites distinguished TNBC from nontransformed breast epithelia and revealed two metabolic subtypes within TNBC that correlate with markers of basal-like versus non-basal-like status. Among the distinguishing metabolites, levels of the cellular redox buffer glutathione were lower in TNBC cell lines compared to controls and markedly lower in non-basal-like TNBC. Significantly, these cell lines showed enhanced sensitivity to pharmacologic inhibition of glutathione biosynthesis that was rescued by N-acetylcysteine, demonstrating a dependence on glutathione production to suppress ROS and support tumor cell survival. Consistent with this, patients whose tumors express elevated levels of γ-glutamylcysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, had significantly poorer survival. We find, further, that agents that limit the availability of glutathione precursors enhance both glutathione depletion and TNBC cell killing by γ-glutamylcysteine ligase inhibitors in vitro Importantly, we demonstrate the ability to this approach to suppress glutathione levels and TNBC xenograft growth in vivo Overall, these findings support the potential of targeting the glutathione biosynthetic pathway as a therapeutic strategy in TNBC and identify the non-basal-like subset as most likely to respond. Mol Cancer Ther; 17(1); 264-75. ©2017 AACR.

Resistance to BET Bromodomain Inhibitors Is Mediated by Kinome Reprogramming in Ovarian Cancer.

Small-molecule BET bromodomain inhibitors (BETis) are actively being pursued in clinical trials for the treatment of a variety of cancers, but the mechanisms of resistance to BETis remain poorly understood. Using a mass spectrometry approach that globally measures kinase signaling at the proteomic level, we evaluated the response of the kinome to targeted BETi treatment in a panel of BRD4-dependent ovarian carcinoma (OC) cell lines. Despite initial inhibitory effects of BETi, OC cells acquired resistance following sustained treatment with the BETi JQ1. Through application of multiplexed inhibitor beads (MIBs) and mass spectrometry, we demonstrate that BETi resistance is mediated by adaptive kinome reprogramming, where activation of compensatory pro-survival kinase networks overcomes BET protein inhibition. Furthermore, drug combinations blocking these kinases may prevent or delay the development of drug resistance and enhance the efficacy of BETi therapy.

Pharmacological profiling of kinase dependency in cell lines across triple-negative breast cancer subtypes.

Triple-negative breast cancers (TNBC), negative for estrogen receptor, progesterone receptor, and ERBB2 amplification, are resistant to standard targeted therapies and exhibit a poor prognosis. Furthermore, they are highly heterogeneous with respect to genomic alterations, and common therapeutic targets are lacking though substantial evidence implicates dysregulated kinase signaling. Recently, six subtypes of TNBC were identified based on gene expression and were proposed to predict sensitivity to a variety of therapeutic agents including kinase inhibitors. To test this hypothesis, we screened a large collection of well-characterized, small molecule kinase inhibitors for growth inhibition in a panel of TNBC cell lines representing all six subtypes. Sensitivity to kinase inhibition correlated poorly with TNBC subtype. Instead, unsupervised clustering segregated TNBC cell lines according to clinically relevant features including dependence on epidermal growth factor signaling and mutation of the PTEN tumor suppressor. We further report the discovery of kinase inhibitors with selective toxicity to these groups. Overall, however, TNBC cell lines exhibited diverse sensitivity to kinase inhibition consistent with the lack of common driver mutations in this disease. Although our findings support specific kinase dependencies in subsets of TNBC, they are not associated with gene expression-based subtypes. Instead, we find that mutation status can be an effective predictor of sensitivity to inhibition of particular kinase pathways for subsets of TNBC.

A high-content screening assay for small-molecule modulators of oncogene-induced senescence.

Cellular senescence is a state of stable cell growth arrest. Activation of oncogenes such as RAS in mammalian cells typically triggers cellular senescence. Oncogene-induced senescence (OIS) is an important tumor suppression mechanism, and suppression of OIS contributes to cell transformation. Oncogenes trigger senescence through a multitude of incompletely understood downstream signaling events that frequently involve protein kinases. To identify target proteins required for RAS-induced senescence, we developed a small-molecule screen in primary human fibroblasts undergoing senescence induced by oncogenic RAS (H-Ras(G12V)). Using a high-content imaging system to monitor two hallmarks of senescence, senescence-associated ß-galactosidase activity expression and inhibition of proliferation, we screened a library of known small-molecule kinase inhibitors for those that suppressed OIS. Identified compounds were subsequently validated and confirmed using a third marker of senescence, senescence-associated heterochromatin foci. In summary, we have established a novel high-content screening platform that may be useful for elucidating signaling pathways mediating OIS by targeting critical pathway components.

Relationship of spindle assembly checkpoint fidelity to species body mass, lifespan, and developmental rate.

We have examined the tolerance of the spindle assembly checkpoint (SAC), as measured by the appearance of tetraploid cells in the presence of a microtubule inhibitor, in a series of primary cell strains derived from species with diverse lifespan and body size. We find that the integrity of the SAC varies among these species. There is a robust correlation between the integrity of the SAC and body size, but poor correlation with longevity and parameters of species development (i.e., time of female fertility, gestation length, and postnatal growth rate). The results suggest that fidelity of the SAC co-evolved more closely with the number of mitoses needed to reach adulthood than with species lifespan.

53BP1 contributes to a robust genomic stability in human fibroblasts.

Faithful repair of damaged DNA is a crucial process in maintaining cell viability and function. A multitude of factors and pathways guides this process and includes repair proteins and cell cycle checkpoint factors. Differences in the maintenance of genomic processes are one feature that may contribute to species-specific differences in lifespan. We predicted that 53BP1, a key transducer of the DNA damage response and cell cycle checkpoint control, is highly involved in maintaining genomic stability and may function differently in cells from different species. We demonstrate a difference in the levels and recruitment of 53BP1 in mouse and human cells following DNA damage. In addition, we show that unresolved DNA damage persists more in mouse cells than in human cells, as evidenced by increased numbers of micronuclei. The difference in micronuclei seems to be related to the levels of 53BP1 present in cells. Finally, we present evidence that unresolved DNA damage correlates with species lifespan. Taken together, these studies suggest a link between recruitment of 53BP1, resolution of DNA damage, and increased species lifespan.

Ku80 facilitates chromatin binding of the telomere binding protein, TRF2.

The Ku70/80 heterodimer is central to non-homologous end joining repair of DNA double-strand breaks and the Ku80 gene appears to be essential for human but not rodent cell survival. The Ku70/80 heterodimer is located at telomeres but its precise function in telomere maintenance is not known. In order to examine the role of Ku80 beyond DNA repair in more detail, we have taken a knockdown approach using a human fibroblast strain. Following targeted Ku80 knockdown, telomere defects are observed and the steady state levels of the TRF2 protein are reduced. Inhibitor studies indicate that this loss of TRF2 is mediated by the proteasome and degradation of TRF2 following Ku depletion appears to involve a decrease in chromatin binding of TRF2, suggesting that the Ku heterodimer enhances TRF2 chromatin association and that non-chromatin bound TRF2 is targeted to the proteasome.