SUMMARY Small molecule BET bromodomain inhibitors (BETi) are actively being pursued in clinical trials for the treatment of a variety of cancers, however, the mechanisms of resistance to BETi 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.

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 Importantly, we demonstrate the ability to this approach to suppress glutathione levels and TNBC xenograft growth 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. .

Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an “autophosphorylation complex.” We developed and applied a structural bioinformatics method to identify all such autophosphorylation kinase complexes in X-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which 5 complexes had not previously been described in the publications describing the crystal structures. These 5 consist of tyrosine residues in the N-terminal juxtamembrane regions of colony stimulating factor 1 receptor (CSF1R, Tyr561) and EPH receptor A2 (EPHA2, Tyr594), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr394) and insulin-like growth factor 1 receptor (IGF1R, Tyr1166), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser142). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro447 to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets.

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.

Triple-negative breast cancers (TNBC), negative for estrogen receptor, progesterone receptor, and Her2 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. While 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.