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The maintenance of redox and metabolic homeostasis is integral to embryonic development. Nuclear factor erythroid 2-related factor 2 (NRF2) is a stress-induced transcription factor that plays a central role in the regulation of redox balance and cellular metabolism. Under homeostatic conditions, NRF2 is repressed by Kelch-like ECH-associated protein 1 (KEAP1). Here, we demonstrate that Keap1 deficiency induces Nrf2 activation and postdevelopmental lethality. Loss of viability is preceded by severe liver abnormalities characterized by an accumulation of lysosomes. Mechanistically, we demonstrate that loss of Keap1 promotes aberrant activation of transcription factor EB (TFEB)/transcription factor binding to IGHM Enhancer 3 (TFE3)-dependent lysosomal biogenesis. Importantly, we find that NRF2-dependent regulation of lysosomal biogenesis is cell autonomous and evolutionarily conserved. These studies identify a role for the KEAP1–NRF2 pathway in the regulation of lysosomal biogenesis and suggest that maintenance of lysosomal homeostasis is required during embryonic development.

Cancer cell metabolism is increasingly recognized as providing an exciting therapeutic opportunity. However, a drug that directly couples targeting of a metabolic dependency with the induction of cell death in cancer cells has largely remained elusive. Here we report that the drug-like small-molecule ironomycin reduces the mitochondrial iron load, resulting in the potent disruption of mitochondrial metabolism. Ironomycin promotes the recruitment and activation of BAX/BAK, but the resulting mitochondrial outer membrane permeabilization (MOMP) does not lead to potent activation of the apoptotic caspases, nor is the ensuing cell death prevented by inhibiting the previously established pathways of programmed cell death. Consistent with the fact that ironomycin and BH3 mimetics induce MOMP through independent nonredundant pathways, we find that ironomycin exhibits marked in vitro and in vivo synergy with venetoclax and overcomes venetoclax resistance in primary patient samples.Significance:Ironomycin couples targeting of cellular metabolism with cell death by reducing mitochondrial iron, resulting in the alteration of mitochondrial metabolism and the activation of BAX/BAK. Ironomycin induces MOMP through a different mechanism to BH3 mimetics, and consequently combination therapy has marked synergy in cancers such as acute myeloid leukemia.This article is highlighted in the In This Issue feature, p. 587

Supplementary movie from Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death

Supplementary table from Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death

Supplementary Data from Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death

Supplementary Figure from Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death

Supplementary Figure S1. Chemotherapy exposure stimulates an increase in pyrimidine nucleotides in TNBC cells.

Cancer cell metabolism is increasingly recognized as providing an exciting therapeutic opportunity. However, a drug that directly couples targeting of a metabolic dependency with the induction of cell death in cancer cells has largely remained elusive. Here we report that the drug-like small-molecule ironomycin reduces the mitochondrial iron load, resulting in the potent disruption of mitochondrial metabolism. Ironomycin promotes the recruitment and activation of BAX/BAK, but the resulting mitochondrial outer membrane permeabilization (MOMP) does not lead to potent activation of the apoptotic caspases, nor is the ensuing cell death prevented by inhibiting the previously established pathways of programmed cell death. Consistent with the fact that ironomycin and BH3 mimetics induce MOMP through independent nonredundant pathways, we find that ironomycin exhibits marked in vitro and in vivo synergy with venetoclax and overcomes venetoclax resistance in primary patient samples. Significance: Ironomycin couples targeting of cellular metabolism with cell death by reducing mitochondrial iron, resulting in the alteration of mitochondrial metabolism and the activation of BAX/BAK. Ironomycin induces MOMP through a different mechanism to BH3 mimetics, and consequently combination therapy has marked synergy in cancers such as acute myeloid leukemia. This article is highlighted in the In This Issue feature, p. 587

Glutamine is a conditionally essential nutrient for many cancer cells, but it remains unclear how consuming glutamine in excess of growth requirements confers greater fitness to glutamine-addicted cancers. By contrasting two breast cancer subtypes with distinct glutamine dependencies, we show that glutamine-indispensable triple-negative breast cancer (TNBC) cells rely on a non-canonical glutamine-to-glutamate overflow, with glutamine carbon routed once through the TCA cycle. Importantly, this single-pass glutaminolysis increases TCA cycle fluxes and replenishes TCA cycle intermediates in TNBC cells, a process that achieves net oxidation of glucose but not glutamine. The coupling of glucose and glutamine catabolism appears hard-wired via a distinct TNBC gene expression profile biased to strip and then sequester glutamine nitrogen, but hampers the ability of TNBC cells to oxidise glucose when glutamine is limiting. Our results provide a new understanding of how metabolically rigid TNBC cells are sensitive to glutamine deprivation and a way to select vulnerable TNBC subtypes that may be responsive to metabolic-targeted therapies.

Adenosine 5'‐monophosphate (AMP)‐activated protein kinase (AMPK) is an important cellular metabolite‐sensing enzyme that can directly sense changes not only in ATP but also in metabolites associated with carbohydrates and fatty acids. However, less is known about whether and how AMPK senses variations in cellular amino acids. Here, we show that cysteine deficiency significantly triggers calcium/calmodulin‐dependent protein kinase kinase 2 (CaMKK2)‐mediated activation of AMPK. In addition, we found that CaMKK2 directly associates with cysteinyl‐tRNA synthetase (CARS), which then binds to AMPKγ2 under cysteine deficiency to activate AMPK. Interestingly, we discovered that cysteine inhibits the binding of CARS to AMPKγ2, and thus, under cysteine deficiency conditions wherein the inhibitory effect of cysteine is abrogated, CARS mediates the binding of AMPK to CaMKK2, resulting in the phosphorylation and activation of AMPK by CaMKK2. Importantly, we demonstrate that blocking AMPK activation leads to cell death under cysteine‐deficient conditions. In summary, our study is the first to show that CARS senses the absence of cysteine and activates AMPK through the cysteine–CARS–CaMKK2–AMPKγ2 axis, a novel adaptation strategy for cell survival under nutrient deprivation conditions.

The coordinated regulation of growth control and metabolic pathways is required to meet the energetic and biosynthetic demands associated with proliferation. Emerging evidence suggests that the Hippo pathway effector Yes-associated protein 1 (YAP) reprograms cellular metabolism to meet the anabolic demands of growth, although the mechanisms involved are poorly understood. Here, we demonstrate that YAP co-opts the sterol regulatory element-binding protein (SREBP)-dependent lipogenic program to facilitate proliferation and tissue growth. Mechanistically, YAP stimulates de novo lipogenesis via mechanistic target of rapamcyin (mTOR) complex 1 (mTORC1) signaling and subsequent activation of SREBP. Importantly, YAP-dependent regulation of serum- and glucocorticoid-regulated kinase 1 (SGK1) is required to activate mTORC1/SREBP and stimulate de novo lipogenesis. We also find that the SREBP target genes fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) are conditionally required to support YAP-dependent proliferation and tissue growth. These studies reveal that de novo lipogenesis is a metabolic vulnerability that can be targeted to disrupt YAP-dependent proliferation and tissue growth.

Despite significant progress, our understanding of how specific oncogenes transform cells is still limited and likely underestimates the complexity of downstream signalling events. To address this gap, we use mass spectrometry-based chemical proteomics to characterize the global impact of an oncogene on the expressed kinome, and then functionally annotate the regulated kinases. As an example, we identify 63 protein kinases exhibiting altered expression and/or phosphorylation in Src-transformed mammary epithelial cells. An integrated siRNA screen identifies nine kinases, including SGK1, as being essential for Src-induced transformation. Accordingly, we find that Src positively regulates SGK1 expression in triple negative breast cancer cells, which exhibit a prominent signalling network governed by Src family kinases. Furthermore, combined inhibition of Src and SGK1 reduces colony formation and xenograft growth more effectively than either treatment alone. Therefore, this approach not only provides mechanistic insights into oncogenic transformation but also aids the design of improved therapeutic strategies., The systemic understanding of oncogenic kinase signalling is still limited. Here, the authors combine chemical proteomics with functional screens to assess the impact of oncogenic Src on the expressed kinome and identify SGK1 as a critical mediator of Src-induced cell transformation.

Activating FMS-like tyrosine kinase 3 (FLT3) mutations occur in approximately 30% of all acute myeloid leukaemias (AMLs) and are associated with poor prognosis. The limited clinical efficacy of FLT3 inhibitor monotherapy has highlighted the need for alternative therapeutic targets and treatments for FLT3-mutant AML. Using human and murine models of MLL-rearranged AML harbouring FLT3 internal tandem duplication (FLT3-ITD) and primary patient samples, we have demonstrated that FLT3-ITD promotes serine uptake and serine synthesis via transcriptional regulation of neutral amino acid transporters (SLC1A4 and SLC1A5) and genes in the de novo serine synthesis pathway (PHGDH and PSAT1). Mechanistically, dysregulation of serine metabolism in FLT3-mutant AML is dependent on the mTORC1-ATF4 axis, that drives RNA-Pol II occupancy at PHGDH, PSAT1, SLC1A4 and SLC1A5. Genetic or pharmacological inhibition of the de novo serine synthesis pathway selectively inhibited the proliferation of FLT3-ITD AML cells, and this was potentiated by withdrawal of exogenous serine. Purine supplementation effectively rescued the antiproliferative effect of inhibiting de novo serine synthesis, consistent with the idea that serine fuels purine nucleotide synthesis in FLT3-mutant AML. Pharmacological inhibition of the de novo serine synthesis pathway, using the PHGDH inhibitor WQ-2101, sensitises FLT3-mutant AML cells to the standard of care chemotherapy agent cytarabine via exacerbation of DNA damage. Collectively, these data reveal new insights as to how FLT3 mutations reprogram metabolism in AML, and reveal a combination therapy strategy to improve the treatment of FLT3-mutant AML.Statement of SignificanceFLT3 mutations are common in AML and are associated with poor prognosis. We show that FLT3-ITD stimulates serine metabolism, thereby rendering FLT3-ITD leukemias dependent on serine for proliferation and survival. This metabolic dependency can be exploited pharmacologically to sensitize FLT3-mutant AML to chemotherapy.

N-functionalized amino acids are important building blocks for the preparation of diverse bioactive molecules including peptides. The development of sustainable manufacturing routes to chiral N-alkylated amino acids remains a significant challenge in the pharmaceutical and fine chemical industries. Herein we report the discovery of a structurally diverse panel of biocatalysts which catalyze the asymmetric synthesis of N-alkyl amino acids via the reductive coupling of ketones and amines. Reactions have been performed on a gram scale to yield optically pure N-alkyl functionalized products in high yields. Postprint

Although the BCL2 inhibitor venetoclax have been transformative in the management of AML, therapeutic resistance and relapse are frequently observed. In light of the urgent need to uncover novel therapeutic options in AML, we sought to study the potential role of ironomycin (AM5), a recently described small molecule that induces cell death through the sequestration of lysosomal iron. To evaluate the effects of ironomycin in AML, we chose a diverse panel of AML cell lines. These data showed a potent and dose-dependent effect, on proliferation, cell cycle progression and survival at a nanomolar range. In contrast to venetoclax, the cell death induced by ironomycin did not result in potent caspase activation or PARP1 cleavage. Neither the caspase inhibitor Z-VAD-fmk nor the necroptosis inhibitor necrostatin-1 did prevent cell death. Consistent with previous observations, we found that ironomycin accumulates in the lysosomes of AML cells leading to a sequestration of iron in this organelle but inhibitors of canonical ferroptosis, including ferrostatin-1 and liproxstatin-1 failed to prevent the activity of ironomycin. To gain greater insight into the molecular mechanism of ironomycin in AML cells, we performed a genome-wide positive-selection resistance screen under ironomycin selection pressure and collected several samples for sequencing. We found nine genes whose knock out conferred resistance to the drug. Interestingly, these data implicated key components of mitochondrial metabolic pathways, including phosphoglycolate phosphatase (PGP), a central phosphatase involved in glycolysis and pentose phosphate pathway (PPP) regulation and Hexokinase 2 (HK2), the first enzyme of glycolysis. Mass-spectrometry metabolomics analyses highlighted that ironomycin treatment significantly reduced key components of the TCA cycle and consequently the reducing agent nicotinamide adenine dinucleotide (NADH) and increased the intracellular concentration of amino acids. These data were corroborated with RNAseq showing a mitochondrial stress response mediated through the Activating Transcription Factor 4 (ATF4) and its paralog Activating Transcription Factor 5 (ATF5). As mitochondria are major hubs of iron utilization for oxidative respiration, we used Mass-spectrometry to measure mitochondrial iron load. We observed a rapid and dose-dependent decrease in mitochondrial iron after treatment mirroring the iron sequestration into the lysosomes and inducing the mitochondrial dysfunction. We next examined the ultrastructural appearance of mitochondria after ironomycin using transmission electron microscopy and observed a dramatic alteration of the structural integrity of mitochondria resulting in abnormal cristae, matrix density changes and mitochondrial membrane blebbing. In cells lacking BAX and BAK, the two main effectors of mitochondrial membrane permeabilization, structural changes and cell death were almost completely rescued but cell proliferation was still markedly affected, consistent with a BAX/BAK dependent cell death following mitochondrial iron deprivation. In vivo imaging confirmed that BAX activation occurred after 30 hours of treatment and preceded cell death, but we observed some major differences with canonical apoptosis induced by venetoclax. First, the structural alterations were clearly distinct. Next, delay between MOMP and cell death was significantly longer and caspase inhibitors weakly delayed cell death. Finally, BCL2 overexpression and P53 deletion did not rescue ironomycin cell death. These non-canonical features prompted us to assess the efficacy of the combination between ironomycin and venetoclax. In vitro experiment on AML cell lines found a high synergy between the two drugs. In vivo experiments on xenotransplanted mice confirmed the efficacy of the combination, which was associated with a significant increase in mice survival in comparison with the controls (Figure). Finally, primary AML samples from patients clinically resistant or refractory to venetoclax were sensitive to ironomycin in monotherapy and even more in combination with venetoclax. These results demonstrate that the novel mechanism of ironomycin action can be leveraged to resensitize AML cells to venetoclax and substitute for cytotoxic drugs as a more effective therapeutic combination in the salvage setting. Figure 1 Figure 1. Disclosures Huang: The Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax. Wei: Novartis, Celgene, AbbVie, Servier, AstraZeneca, and Amgen: Research Funding; Novartis, Janssen, Amgen, Roche, Pfizer, Abbvie, Servier, BMS, Macrogenics, Agios, Gilead: Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria.

Chemotherapy resistance is a major barrier to the treatment of triple-negative breast cancer (TNBC), and strategies to circumvent resistance are required. Using in vitro and in vivo metabolic profiling of TNBC cells, we show that an increase in the abundance of pyrimidine nucleotides occurs in response to chemotherapy exposure. Mechanistically, elevation of pyrimidine nucleotides induced by chemotherapy is dependent on increased activity of the de novo pyrimidine synthesis pathway. Pharmacologic inhibition of de novo pyrimidine synthesis sensitizes TNBC cells to genotoxic chemotherapy agents by exacerbating DNA damage. Moreover, combined treatment with doxorubicin and leflunomide, a clinically approved inhibitor of the de novo pyrimidine synthesis pathway, induces regression of TNBC xenografts. Thus, the increase in pyrimidine nucleotide levels observed following chemotherapy exposure represents a metabolic vulnerability that can be exploited to enhance the efficacy of chemotherapy for the treatment of TNBC. Significance: The prognosis for patients with TNBC with residual disease after chemotherapy is poor. We find that chemotherapy agents induce adaptive reprogramming of de novo pyrimidine synthesis and show that this response can be exploited pharmacologically, using clinically approved inhibitors of de novo pyrimidine synthesis, to sensitize TNBC cells to chemotherapy. Cancer Discov; 7(4); 391–9. ©2017 AACR. See related article by Mathur et al., p. 380. This article is highlighted in the In This Issue feature, p. 339

Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 30% of all acute myeloid leukaemias (AMLs) and are associated with poor prognosis. The clinical utility of FLT3 inhibitor monotherapy has been limited by the rapid development of resistance, highlighting the need for identification of alternative therapeutic targets for the treatment of FLT3-mutant AML. Using a syngeneic murine model of AML harbouring FLT3 internal tandem duplication (FLT3-ITD), we demonstrate that FLT3-ITD promotes serine uptake and serine synthesis via transcriptional regulation of neutral amino acid transporters (SLC1A4 and SLC1A5) and genes in the de novo serine synthesis pathway (PHGDH and PSAT1). Mechanistically, dysregulation of serine metabolism was dependent on the mTORC1-ATF4 axis, which promoted RNA-Pol II occupancy at PHGDH, PSAT1, SLC1A4 and SLC1A5 in AML cells harbouring FLT3-ITD mutations. Genetic or pharmacological inhibition of the de novo serine synthesis pathway selectively inhibited the proliferation and viability of FLT3-ITD AML cells, and purine supplementation effectively rescued the apoptotic effects of inhibiting FLT3-ITD, consistent with the hypothesis that serine fuels purine nucleotide synthesis in FLT3-ITD AML cells. To exploit these findings in the context of standard-of-care therapy in AML, we show that pharmacological inhibition of the de novo serine synthesis pathway, using the PHGDH inhibitor WQ-2101, sensitises FLT3-ITD AML cells to the chemotherapy agent cytarabine by greatly exacerbating the DNA damage response in AML cell lines harbouring FLT3-ITD mutations, primary AML patient samples, and an aggressive PDX mouse model of FLT3-ITD-driven AML. Collectively, these data reveal new insights into FLT3-ITD-induced metabolic reprogramming in AML, and suggest a novel combinatorial therapeutic strategy for the treatment of FLT3-mutant AML.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer, originating from liver cells known as hepatocytes. At the molecular level, HCC is driven by transcription factors which are able to reprogram metabolism to support tumorigenesis that are still poorly understood. The Yes-associated protein (Yap) is the nuclear effector of the Hippo pathway, responsible in regulating organ size control and metabolism. Metabolic reprogramming has recently emerged as a fundamental hallmark of cancer. In our previous studies, we found that Yap integrates the anabolic demands of tumour growth by reprogramming glutamine and glucose metabolism to support nucleotide biosynthesis. The central aim of this study was to determine the role that Yap plays in regulating lipid metabolism in the context of liver cancer. We took advantage of a larval zebrafish model in which a hyperactivated form of Yap is specifically expressed in hepatocytes (lf:YapS87A;lf:NLS-mcherry). We found that the expression of Yap was sufficient to stimulate de novo lipogenesis (DNL) and induce lipid droplet formation in hepatocytes (steatosis). To determine whether the stimulation of DNL was required for oncogenic growth, we exposed larvae to pharmacological inhibitors of DNL and examined the impact on growth at the cellular level by multiphoton microscopy. Strikingly, we identified that fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD) inhibitors suppressed Yap-driven growth. To complement these studies, we took a genetic approach using CRISPR to generate FAS and SCD KO zebrafish larvae and we found that Yap-driven growth required both FAS and SCD. Importantly both chemical and genetic suppression of FAS and SCD activity had no effect on normal liver growth. Together, these findings suggest that oncogenic Yap-driven growth is conditionally dependent upon the stimulation of DNL. Consequently, these studies provide a rationale for examining the clinical efficacy of DNL inhibitors to combat liver cancer. Citation Format: Talhah M. Salmi, Srimayee Vaidyanathan, Malcolm J. McConville, Kristin K. Brown, Andrew G. Cox. Yap reprograms de novo lipogenesis to fuel liver cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-015.

The Hippo pathway and its nuclear effector Yap regulate organ size and cancer formation. While many modulators of Hippo activity have been identified, little is known about the Yap target genes that mediate these growth effects. Here, we show that yap−/− mutant zebrafish exhibit defects in hepatic progenitor potential and liver growth due to impaired glucose transport and nucleotide biosynthesis. Transcriptomic and metabolomic analyses reveal that Yap regulates expression of glucose transporter glut1, causing decreased glucose uptake and use for nucleotide biosynthesis in yap−/− mutants, and impaired glucose tolerance in adults. Nucleotide supplementation improves Yap deficiency phenotypes, indicating functional importance of glucose-fueled nucleotide biosynthesis. Yap-regulated glut1 expression and glucose uptake are conserved in mammals, suggesting that stimulation of anabolic glucose metabolism is an evolutionarily conserved mechanism by which the Hippo pathway controls organ growth. Together, our results reveal a central role for Hippo signaling in glucose metabolic homeostasis., National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant P30DK034854), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant 1R01DK090311), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant R24OD017870), National Institutes of Health (U.S.) (Grant P30DK034854), National Institutes of Health (U.S.) (Grant 1R01DK090311), National Institutes of Health (U.S.) (Grant 1R01DK105198), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant 1R01DK105198), National Institutes of Health (U.S.) (Grant R24OD017870), National Institute of General Medical Sciences (NIGMS) (Grant T32GM007753), NHMRC (Grant 1146558), National Cancer Institute (U.S.) (Grant 5P01CA120964), National Cancer Institute (U.S.) (Grant 5P30CA006516)

The assessment of the suitability of novel targets to intervention by different modalities, e.g. small molecules or antibodies, is increasingly seen as important in helping to select the most progressable targets at the outset of a drug discovery project. This perspective considers differing aspects of tractability and how it can be assessed using in silico and experimental approaches. We also share some of our experiences in using these approaches.

Summary Toxic liver injury is a leading cause of liver failure and death because of the organ's inability to regenerate amidst massive cell death, and few therapeutic options exist. The mechanisms coordinating damage protection and repair are poorly understood. Here, we show that S-nitrosothiols regulate liver growth during development and after injury in vivo; in zebrafish, nitric-oxide (NO) enhanced liver formation independently of cGMP-mediated vasoactive effects. After acetaminophen (APAP) exposure, inhibition of the enzymatic regulator S-nitrosoglutathione reductase (GSNOR) minimized toxic liver damage, increased cell proliferation, and improved survival through sustained activation of the cytoprotective Nrf2 pathway. Preclinical studies of APAP injury in GSNOR-deficient mice confirmed conservation of hepatoprotective properties of S-nitrosothiol signaling across vertebrates; a GSNOR-specific inhibitor improved liver histology and acted with the approved therapy N-acetylcysteine to expand the therapeutic time window and improve outcome. These studies demonstrate that GSNOR inhibitors will be beneficial therapeutic candidates for treating liver injury.

© 2016, National Academy of Sciences. All rights reserved. Selenium, an essential micronutrient known for its cancer prevention properties, is incorporated into a class of selenocysteine-containing proteins (selenoproteins). Selenoprotein H (SepH) is a recently identified nucleolar oxidoreductase whose function is not well understood. Here we report that seph is an essential gene regulating organ development in zebrafish. Metabolite profiling by targeted LC-MS/MS demonstrated that SepH deficiency impairs redox balance by reducing the levels of ascorbate and methionine, while increasing methionine sulfoxide. Transcriptome analysis revealed that SepH deficiency induces an inflammatory response and activates the p53 pathway. Consequently, loss of seph renders larvae susceptible to oxidative stress and DNA damage. Finally, we demonstrate that seph interacts with p53 deficiency in adulthood to accelerate gastrointestinal tumor development. Overall, our findings establish that seph regulates redox homeostasis and suppresses DNA damage. We hypothesize that SepH deficiency may contribute to the increased cancer risk observed in cohorts with low selenium levels.

Breast carcinoma is the leading cause of cancer-related mortality in women worldwide with an estimated 1.38 million new cases and 458,000 deaths in 2008 alone1. This malignancy represents a heterogeneous group of tumours with characteristic molecular features, prognosis, and responses to available therapy2–4. Recurrent somatic alterations in breast cancer have been described including mutations and copy number alterations, notably ERBB2 amplifications, the first successful therapy target defined by a genomic aberration5. Prior DNA sequencing studies of breast cancer genomes have revealed additional candidate mutations and gene rearrangements 6–10. Here we report the whole-exome sequences of DNA from 103 human breast cancers of diverse subtypes from patients in Mexico and Vietnam compared to matched-normal DNA, together with whole-genome sequences of 22 breast cancer/normal pairs. Beyond confirming recurrent somatic mutations in PIK3CA11, TP536, AKT112, GATA313, and MAP3K110, we discovered recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1. Furthermore, we have identified a recurrent MAGI3-AKT3 fusion enriched in triple-negative breast cancer lacking estrogen and progesterone receptors and ERBB2 expression. The Magi3-Akt3 fusion leads to constitutive activation of Akt kinase, which is abolished by treatment with an ATP-competitive Akt small-molecule inhibitor.

Isothiocyanates are a class of phytochemicals with widely reported anti-cancer and anti-inflammatory activity. However, knowledge of their activity at a molecular level is limited. The objective of this study was to identify biological targets of phenethyl isothiocyanate (PEITC) using an affinity purification approach. An analogue of PEITC was synthesized to enable conjugation to a solid-phase resin. The pleiotropic cytokine macrophage migration inhibitory factor (MIF) was the major protein captured from cell lysates. Site-directed mutagenesis and mass spectrometry showed that PEITC covalently modified the N-terminal proline residue of MIF. This resulted in complete loss of catalytic tautomerase activity and disruption of protein conformation, as determined by impaired recognition by a monoclonal antibody directed to the region that receptors and interacting proteins bind to MIF. The conformational change was supported by in silico modeling. Monoclonal antibody binding to plasma MIF was disrupted in humans consuming watercress, a major dietary source of PEITC. The isothiocyanates have significant potential for development as MIF inhibitors, and this activity may contribute to the biological properties of these phytochemicals.

The Hippo pathway is an important regulator of organ size and tumorigenesis. It is unclear, however, how Hippo signalling provides the cellular building blocks required for rapid growth. Here, we demonstrate that transgenic zebrafish expressing an activated form of the Hippo pathway effector Yap1 (also known as YAP) develop enlarged livers and are prone to liver tumour formation. Transcriptomic and metabolomic profiling identify that Yap1 reprograms glutamine metabolism. Yap1 directly enhances glutamine synthetase (glul) expression and activity, elevating steady-state levels of glutamine and enhancing the relative isotopic enrichment of nitrogen during de novo purine and pyrimidine biosynthesis. Genetic or pharmacological inhibition of GLUL diminishes the isotopic enrichment of nitrogen into nucleotides, suppressing hepatomegaly and the growth of liver cancer cells. Consequently, Yap-driven liver growth is susceptible to nucleotide inhibition. Together, our findings demonstrate that Yap1 integrates the anabolic demands of tissue growth during development and tumorigenesis by reprogramming nitrogen metabolism to stimulate nucleotide biosynthesis.

The phosphoinositide 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling network is a master regulator of processes that contribute to tumorigenesis and tumor maintenance. The PI3K pathway also plays a critical role in driving resistance to diverse anti-cancer therapies. This review article focuses on mechanisms by which the PI3K pathway contributes to therapy resistance in cancer, and highlights potential combination therapy strategies to circumvent resistance driven by PI3K signaling. In addition, resistance mechanisms that limit the clinical efficacy of small molecule inhibitors of the PI3K pathway are discussed.

Nucleotide-binding domain leucine-rich repeat proteins (NLRs) play a key role in immunity and disease through their ability to modulate inflammation in response to pathogen-derived and endogenous danger signals. Here, we identify the requirements for activation of NLRP1, an NLR protein associated with a number of human pathologies, including vitiligo, rheumatoid arthritis, and Crohn disease. We demonstrate that NLRP1 activity is dependent upon ASC, which associates with the C-terminal CARD domain of NLRP1. In addition, we show that NLRP1 activity is dependent upon autolytic cleavage at Ser(1213) within the FIIND. Importantly, this post translational event is dependent upon the highly conserved distal residue His(1186). A disease-associated single nucleotide polymorphism near His(1186) and a naturally occurring mRNA splice variant lacking exon 14 differentially affect this autolytic processing and subsequent NLRP1 activity. These results describe key molecular pathways that regulate NLRP1 activity and offer insight on how small sequence variations in NLR genes may influence human disease pathogenesis.

SummaryIntegrin recycling is critical for cell migration. Protein kinase D (PKD) mediates signals from the platelet-derived growth factor receptor (PDGF-R) to control αvβ3 integrin recycling. We now show that Rabaptin-5, a Rab5 effector in endosomal membrane fusion, is a PKD substrate. PKD phosphorylates Rabaptin-5 at Ser407, and this is both necessary and sufficient for PDGF-dependent short-loop recycling of αvβ3, which in turn inhibits α5β1 integrin recycling. Rab4, but not Rab5, interacts with phosphorylated Rabaptin-5 toward the front of migrating cells to promote delivery of αvβ3 to the leading edge, thereby driving persistent cell motility and invasion that is dependent on this integrin. Consistently, disruption of Rabaptin-5 Ser407 phosphorylation reduces persistent cell migration in 2D and αvβ3-dependent invasion. Conversely, invasive migration that is dependent on α5β1 integrin is promoted by disrupting Rabaptin phosphorylation. These findings demonstrate that the PKD pathway couples receptor tyrosine kinase signaling to an integrin switch via Rabaptin-5 phosphorylation.

Hepatocellular carcinoma is a global health problem with poor prognosis and limited therapeutic options. Recently, the Hippo pathway has emerged as a master regulator of organ size control and tumorigenesis. However, the metabolic impact of the pathway is poorly understood. Using a transgenic zebrafish model with liver-specific activation of the Hippo pathway effector Yap, we have shown that Yap promotes hepatomegaly and liver dysplasia. In addition, we demonstrate that the Yap transgenics are highly susceptible to chemically-induced hepatocarcinogenesis. Transcriptomic and metabolomic profiling reveals that Yap enhances glutamine synthase (GS) expression and elevates steady-state levels of glutamine, respectively. Intervention studies with the GS inhibitor methionine sulfoximine established that elevated GS activity contributes to the rapid liver growth observed during Yap-driven hepatomegaly. Finally, studies in cultured human cancer cells identify GS as a bone-fide Yap target gene, confirming that the GS regulation by Yap is evolutionarily conserved. We conclude that Yap regulates GS expression and reprograms nitrogen metabolism, which contributes to liver growth during development and tumorigenesis. We hypothesize that Yap integrates the anabolic demands of rapid cell proliferation by increasing the flux of glutamine into nucleotide biosynthesis. Citation Format: Andrew G. Cox, Katie L. Hwang, Kimberley Evason, Kristin K. Brown, Sebastian Beltz, Keelin O'Connor, Giorgio G. Galli, Dean Yimlamai, Sagar Chhangawala, Evan Lien, Fernando D. Camargo, John Asara, Yariv Houvras, Didier Y. Stainier, Wolfram Goessling. Yap reprograms glutamine metabolism and supports growth during liver development and tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5151. doi:10.1158/1538-7445.AM2015-5151

Resistance to cytotoxic chemotherapy agents is a common phenomenon in breast cancer and has a drastic impact on patient survival. Characterization of the mechanisms that contribute to chemotherapy resistance, and identification of strategies to circumvent resistance, would provide more effective therapies for the management of breast cancer. In this study we show that in addition to inflicting DNA damage, the cytotoxic chemotherapy agent doxorubicin triggers rapid and sustained activation of Akt in breast cancer cells. Pharmacological inhibitors of PI 3-K or Akt effectively block Akt signaling downstream of DNA damage and render cells markedly more sensitive to doxorubicin. We sought to identify mechanisms by which Akt promotes resistance to doxorubicin and have identified MERIT40, a component of the nuclear BRCA1-A DNA damage repair complex, as a novel Akt substrate that is phosphorylated when cells are exposed to doxorubicin. We have demonstrated that MERIT40 phosphorylation is necessary for BRCA1-A complex assembly and is required for resolution of DNA damage. We propose that inhibition of MERIT40 phosphorylation contributes to the efficacy of combination therapy with PI 3-K or Akt inhibitors and doxorubicin. We also show that MERIT40 phosphorylation is associated with ER status in breast tumor tissue samples, suggesting that MERIT40 phosphorylation could be used as a biomarker in ER-positive breast cancer patients to predict sensitivity to cytotoxic chemotherapy agents. Our data also indicates that patients with high levels of phosphorylated MERIT40 would likely benefit from combination therapy with Akt inhibitors and doxorubicin. We are currently investigating additional mechanisms by which Akt activation downstream of DNA damage promotes chemotherapy resistance with the aim of identifying novel combination therapy regimens to circumvent chemotherapy resistance. Citation Format: Kristin K. Brown, Laleh Montaser-Kouhsari, Andrew H. Beck, Alex Toker. MERIT40 is an Akt substrate that promotes resolution of DNA damage induced by chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5477. doi:10.1158/1538-7445.AM2015-5477

Isothiocyanates are a class of naturally occuring chemopreventive agents known to be effective at triggering apoptosis. In this study, we show that whereas overexpression of the oncoprotein Bcl-2 renders Jurkat T-lymphoma cells resistant to a range of cytotoxic agents, phenethyl isothiocyanate is able to overcome the inhibitory action of Bcl-2 and trigger apoptosis. A 50-fold increase in Bcl-2 expression shifted the dose-response curve, with an increase in the phenethyl isothiocyanate LD50 from 7 to 15 μmol/L, but there was still a complete loss in cell viability at doses in excess of 20 μmol/L. At these concentrations, cytotoxicity was strongly associated with caspase activation, phosphatidylserine exposure, and morphologic changes characteristic of apoptosis. Cytotoxicity was inhibited by treatment of the cells with a broad-spectrum caspase inhibitor. A structure-activity analysis showed that the phenethyl and benzyl isothiocyanates were most effective at triggering apoptosis in cells overexpressing Bcl-2 whereas phenyl isothiocyanate and benzyl thiocyanate had no proapoptotic activity. Allyl isothiocyanate also had limited efficacy despite its ability to trigger apoptosis in the parental Jurkat cell line. From this information, we propose that isothiocyanates modify a key cysteine residue in an apoptosis regulatory protein and that the aromatic side chain facilitates access to the target site. An in-depth investigation of the cellular targets of the aromatic isothiocyanates is warranted.(Cancer Res 2006; 66(13): 6772-7)

Aberrant activation of the PI3K/Akt pathway is found in approximately 70% of breast cancers. We recently identified a recurrent rearrangement that produces an in-frame fusion protein containing Akt3 and the scaffolding protein MAGI3. MAGI3-Akt3 is the first fusion protein that has been identified in the PI3K pathway. We propose that MAGI3-Akt3 is a novel oncogene in breast cancer and that the oncogenic activity of the fusion protein results from disruption of the PI3K/Akt pathway. Within the fusion, Akt3 has a disrupted PH domain prior to glutamate 17 (E17). As a result the fusion protein is constitutively phosphorylated in the Akt3 kinase domain and expression of MAGI3-Akt3 in breast cancer cells enhances Akt substrate phosphorylation. Constitutive MAGI3-Akt3 kinase activity can be inhibited by ATP-competitive small molecule Akt inhibitors but is resistant to the allosteric Akt inhibitor MK2206 which is currently in phase II clinical trials. Our results indicate that ATP competitive Akt inhibitors should be evaluated in clinical trials for the treatment of fusion-positive breast cancers. The fusion protein also contains a severely truncated portion of the scaffolding protein MAGI3. In particular, the fusion lacks the second PDZ domain of MAGI3 that has been reported to bind to PTEN and be required for the inhibitory effect of PTEN on PI3K pathway activity. We have demonstrated that disruption of MAGI3 in breast cancer cell lines does indeed promote PI3K pathway hyperactivity in a PTEN-dependent manner. In addition, the fusion lacks the fifth PDZ domain of MAGI3 and our studies indicate that this disrupts the interaction of MAGI3 with PHLPP2, a phosphatase that directly dephosphorylates Akt at pS473. Our studies therefore demonstrate that the MAGI3-Akt3 translocation significantly perturbs PI3K pathway activity via activation of an oncogene (Akt3) and dysregulation of two tumor suppressors (PTEN and PHLPP2). We are currently continuing studies to elucidate the oncogenic mechanism(s) of MAGI3-Akt3 in breast cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B195. Citation Format: Kristin Brown, Alex Toker. MAGI3-Akt3, a novel fusion protein in the PI3K pathway in cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B195.

Many cancer cells generate energy by rapidly converting glucose to lactate in the cytosol, a process termed aerobic glycolysis. This metabolic phenotype is recognized as one of the hallmarks of cancer and is enabled by lactate dehydrogenase (LDH), which catalyzes pyruvate to lactate inter-conversion. We find that hepatocellular carcinoma cells express micromolar quantities of LDH5 and that LDH5 protein down-regulation takes about 5 days allowing time for the cells to adapt their metabolism. Since metabolic processes happen in minutes, addressing consequences of LDH5 inhibition by protein down-regulation is inadequate. We screened the GSK compound library and identified a series of quinoline acids as NADH-competitive LDH5 inhibitors. Subsequent lead optimization yielded molecules with LDH5 inhibitory potencies as low as 2-3 nM and selectivity over LDH1 on the order of 10-100-fold. These molecules were cell-permeable and did not have any appreciable activity against a panel of approximately fifty common enzymes, receptors and ion channels, making them the most potent and selective LDH5 inhibitors identified to date. Using these tool inhibitors, we find that rapid chemical inhibition of LDH5 in Snu398 hepatocellular carcinoma cells results in profound inhibition of lactate production and increase in pyruvate as measured by mass spectrometric analysis. Real-time analysis by NMR spectroscopy of live Snu398 cells fed with 13C-labeled glucose demonstrated that chemical LDH5 inhibition led to a rapid decrease in glucose uptake and concomitant slow-down of lactate production. Comprehensive analysis of more than 500 metabolites upon LDH5 inhibition in Snu398 cells revealed that the cytosolic glycolysis pathway was significantly impeded with some up-stream intermediates increasing as much as 40-fold. As the cell lost its ability for cytosolic glucose processing, the TCA cycle activity increased indicating that pyruvate entered the mitochondria and restored their activity resulting in increased oxygen consumption upon LDH5 inhibition. Several pathways that rely on glycolytic and TCA intermediates were also upregulated, including fatty acid metabolism and pentose phosphate pathway. LDH5 inhibition also strongly potentiated PKM2 activity. These profound metabolic alterations greatly impaired cell survival and induced cell death in Snu398 cells. In summary, we have shown that rapid chemical inhibition of LDH5 leads to profound metabolic alterations and impairs cell survival in hepatocellular carcinoma cells making it a compelling strategy for treating solid tumors relying on aerobic glycolysis. Citation Format: Julia Billiard, Roland Annan, Jennifer Ariazi, Jacques Briand, Kristin Brown, Nino Campobasso, Subhas Chakravorty, Deping Chai, Mariela Colón, Elizabeth Davenport, Christopher Dodson, Nathan Gaul, Seth Gilbert, Anthony Jurewicz, Hong Lu, Dean McNulty, Jeanelle McSurdy-Freed, Lisa Miller, Kelvin Nurse, Paru Rao Nuthulaganti, Chad Quinn, Jessica Schneck, Gilbert Scott, Tony Shaw, Christian Sherk, Angela Smallwood, Sharon Sweitzer, James Villa, Gregory Waitt, Richard Wooster, Kevin Duffy. Rapid LDH5 inhibition reverses malignant metabolic phenotype and impairs survival of hepatocellular carcinoma cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5418. doi:10.1158/1538-7445.AM2013-5418

Aberrant activation of the PI3-K/Akt pathway is found in approximately 70% of breast cancers. We recently identified a recurrent rearrangement that produces an in-frame fusion protein containing Akt3 and the scaffolding protein MAGI3 (1). MAGI3-Akt3 is the first fusion protein that has been identified in the PI 3-K pathway. We propose that MAGI3-Akt3 is a novel oncogene in breast cancer and that the oncogenic activity of the fusion protein results from disruption of the PI 3-K/Akt pathway. Within the fusion, Akt3 has a disrupted PH domain prior to glutamate 17 (E17). As a result the fusion protein is constitutively phosphorylated in the Akt3 kinase domain and expression of MAGI3-Akt3 in breast cancer cells enhances Akt substrate phosphorylation. Constitutive MAGI3-Akt3 kinase activity can be inhibited by ATP-competitive small molecule Akt inhibitors but is resistant to the allosteric Akt inhibitor MK2206 which is currently in phase II clinical trials. Our results indicate that ATP competitive Akt inhibitors should be evaluated in clinical trials for the treatment of fusion-positive breast cancers. The fusion protein also contains a severely truncated portion of the scaffolding protein MAGI3. In particular, the fusion lacks the second PDZ domain of MAGI3 that has been reported to bind to PTEN and be required for the inhibitory effect of PTEN on PI 3-K pathway activity. We have demonstrated that disruption of MAGI3 in breast cancer cell lines does indeed promote PI 3-K pathway hyperactivity in a PTEN-dependent manner. In addition, the fusion lacks the fifth PDZ domain of MAGI3 and our studies indicate that this disrupts the interaction of MAGI3 with PHLPP2, a phosphatase that directly dephosphorylates Akt at pS473. Our studies therefore demonstrate that the MAGI3-Akt3 translocation significantly perturbs PI 3-K pathway activity via activation of an oncogene (Akt3) and dysregulation of two tumor suppressors (PTEN and PHLPP2). We are currently continuing studies to elucidate the oncogenic mechanism(s) of MAGI3-Akt3 in breast cancer. Citation Format: Kristin Brown, Alex Toker. MAGI3-Akt3, a novel fusion protein in the PI 3-K pathway in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 549. doi:10.1158/1538-7445.AM2013-549

Today, more than 55% of the world's breast cancer cases are diagnosed in low and middle-income countries and in 2020, more that 70% of the cases will come from the developing nations. In Mexico, breast cancer-specific mortality doubled during the past 20 years, representing the second-leading cause of death in women between 30 and 59 years and the leading cause of cancer related death in the female population. According to statistics, in Mexico a woman dies due to breast cancer every two hours. Even though breast cancer represents a major public health problem in the developing world, knowledge about the genetic and genomic structure of breast tumors in Mexican or Latin American populations is very limited. In the past four years, we have participated in the Slim Initiative of Genomic Medicine (SIGMA) Project, a collaboration between the Carlos Slim Institute of Health, the Broad Institute, and the National Institute of Genomic Medicine in Mexico city. The goal of the SIGMA project is to characterize the genomic basis of common diseases, including several types of cancer. This effort has focused on the application of whole genome and whole exome sequencing of human tumors. In the case of breast cancer, we have analyzed the whole genomes of 22 tumor/normal tissue pairs and the whole exomes of 103 tumor/normal tissues from Mexican and Vietnamese patients. Sequence analysis led to the novel identification of potential loss of function mutations of the CBFB transcription factor, and deletions of its partner RUNX1, an event which has never been previously reported in breast tumors or in any other epithelial tumor. Of clinical relevance, we also identified a somatic translocation involving MAGI3 and AKT3 in a triple negative breast tumor. Ectopic expression of the fusion transcrip leads to constitutive phosphorylation of downstream GSK and loss of contact inhibition. Most importantly, the activity of the fusion protein can be abrogated by an ATP-competitive small molecule inhibitor of AKT, potentially representing a new therapeutic avenue for these patients. In parallel with sequencing, we have also been working on the analysis of somatic DNA copy number aberrations, messenger RNA expression, and microRNA expression patterns in tumors from Mexican patients. Intrinsic breast cancer sub-typing in 125 tumors from Mexican patients showed that 13.6% of the tumors were basal-like, 16.8% were Her2-enriched, 24.8% Luminal A, 34.4% Luminal B and 10.4 normal-like. With microRNA expression, we have identified a group of microRNAs whose role in breast cancer has not been previously described and are currently analyzing differential microRNA expression across tumor sub-types, in particular triple negative tumors, where we have been able to identify at least three different tumor sub-groups based on microRNA expression patterns. Citation Format: Shantanu Banerji, Kristian Cibulskis, Claudia Rangel-Escareño, Kristin K. Brown, Scott L. Carter, Abbie M. Frederick, Michael S. Lawrence, Andrey Y. Sivachenko, Carrie Sougnez, Lihua Zou, Maria L. Cortes, Juan C. Fernandez-Lopez, Shouyong Peng, Kristin G. Ardlie, Daniel Auclair, Veronica Bautista-Piña, Fujiko Duke, Joshua Francis, Joonil Jung, Antonio Maffuz-Aziz, Robert C. Onofrio, Melissa Parkin, Nam H. Pho, Valeria Quintanar-Jurado, Alex H. Ramos, Rosa Rebollar-Vega, Sergio A. Rodríguez-Cuevas, Sandra L. Romero-Cordoba, Steven E. Schumacher, Nicolas Stransky, Kristin M. Thompson, Laura Uribe-Figueroa, Jose Baselga, Rameen Beroukhim, Kornelia Polyak, Dennis C. Sgroi, Andrea L. Richardson, Gerardo Jimenez-Sánchez, Eric S. Lander, Stacey B. Gabriel, Levi A. Garraway, Todd R. Golub, Jorge Meléndez-Zajgla, Alex Toker, Gad Getz, Matthew Meyerson, Alfredo Hidalgo-Miranda. Molecular profiling of breast cancer in Mexico: Identification of novel therapeutic targets through whole genome sequencing analysis. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr PL07-01.

Mitochondrial peroxiredoxin 3 (Prx 3) is rapidly oxidized in cells exposed to phenethyl isothiocyanate (PEITC) and auranofin (AFN), but the mechanism of oxidation is unclear. Using HL-60 cells deplete of mitochondrial DNA we show that peroxiredoxin 3 oxidation and cytotoxicity requires a functional respiratory chain. Thioredoxin reductase (TrxR) could be inhibited by up to 90% by auranofin without direct oxidation of peroxiredoxin 3. However, inhibition of thioredoxin reductase promoted peroxiredoxin 3 oxidation and cytotoxicity in combination with phenethyl isothiocyanate or antimycin A. We conclude that rapid peroxiredoxin 3 oxidation occurs as a consequence of increased oxidant production from the mitochondrial respiratory chain.

SummaryResistance to cytotoxic chemotherapy drugs, including doxorubicin, is a significant obstacle to the effective treatment of breast cancer. Here, we have identified a mechanism by which the PI3K/Akt pathway mediates resistance to doxorubicin. In addition to inducing DNA damage, doxorubicin triggers sustained activation of Akt signaling in breast cancer cells. We show that Akt contributes to chemotherapy resistance such that PI3K or Akt inhibitors sensitize cells to doxorubicin. We identify MERIT40, a component of the BRCA1-A DNA damage repair complex, as an Akt substrate that is phosphorylated following doxorubicin treatment. MERIT40 phosphorylation facilitates assembly of the BRCA1-A complex in response to DNA damage and contributes to DNA repair and cell survival following doxorubicin treatment. Finally, MERIT40 phosphorylation in human breast cancers is associated with estrogen receptor positivity. Our findings suggest that combination therapy with PI3K or Akt inhibitors and doxorubicin may constitute a successful strategy for overcoming chemotherapy resistance.