Ludwig Center at Harvard

Clement, E., Inuzuka, H., Nihira, N., Wei, W. & Toker, A. Skp2-dependent reactivation of AKT drives resistance to PI3K inhibitors. Science Signaling 11, 521, (2018).Abstract

The PI3K-AKT kinase signaling pathway is frequently deregulated in human cancers, particularly breast cancer, where amplification and somatic mutations of PIK3CA occur with high frequency in patients. Numerous small-molecule inhibitors targeting both PI3K and AKT are under clinical evaluation, but dose-limiting toxicities and the emergence of resistance limit therapeutic efficacy. Various resistance mechanisms to PI3K inhibitors have been identified, including de novo mutations, feedback activation of AKT, or cross-talk pathways. We found a previously unknown resistance mechanism to PI3K pathway inhibition that results in AKT rebound activation. In a subset of triple-negative breast cancer cell lines, treatment with a PI3K inhibitor or depletion of PIK3CA expression ultimately promoted AKT reactivation in a manner dependent on the E3 ubiquitin ligase Skp2, the kinases IGF-1R (insulin-like growth factor 1 receptor) and PDK-1 (phosphoinositide-dependent kinase-1), and the cell growth and metabolism-regulating complex mTORC2 (mechanistic target of rapamycin complex 2), but was independent of PI3K activity or PIP₃ production. Resistance to PI3K inhibitors correlated with the increased abundance of Skp2, ubiquitylation of AKT, cell proliferation in culture, and xenograft tumor growth in mice. These findings reveal a ubiquitin signaling feedback mechanism by which PI3K inhibitor resistance may emerge in aggressive breast cancer cells.

Liu, H., et al. Identifying and Targeting Sporadic Oncogenic Genetic Aberrations in Mouse Models of Triple Negative Breast Cancer. Cancer Discov (2017). Publisher's Version Abstract

Triple negative breast cancers (TNBC) are genetically characterized by aberrations in TP53 and a low rate of activating point mutations in common oncogenes, rendering it challenging in applying targeted therapies. We performed whole exome sequencing (WES) and RNAseq to identify somatic genetic alterations in mouse models of TNBCs driven by loss of Trp53 alone or in combination with Brca1. Amplifications or translocations that resulted in elevated oncoprotein expressions or oncoprotein-containing fusions, respectively, as well as frame-shift mutations of tumor suppressors were identified in approximately 50% of the tumors evaluated. While the spectrum of sporadic genetic alterations was diverse, the majority had in common the ability to activate the MAPK/PI3K pathways. Importantly, we demonstrated that approved or experimental drugs efficiently induce tumor regression specifically in tumors harboring somatic aberrations of the drug target. Our study suggests that the combination of WES and RNAseq on human TNBC will lead to the identification of actionable therapeutic targets for precision medicine guided TNBC treatment.

Lien, E.C., Ghisolfi, L., Geck, R.C., Asara, J.M. & Toker, A. Oncogenic PI3K promotes methionine dependency in breast cancer cells through the cystine-glutamate antiporter xCT. Science Signaling 10, 510, (2017). Publisher's Version Abstract

The precursor homocysteine is metabolized either through the methionine cycle to produce methionine or through the transsulfuration pathway to synthesize cysteine. Alternatively, cysteine can be obtained through uptake of its oxidized form, cystine. Many cancer cells exhibit methionine dependency such that their proliferation is impaired in growth media in which methionine is replaced by homocysteine. We showed that oncogenic PIK3CA and decreased expression of SLC7A11, a gene that encodes a cystine transporter also known as xCT, correlated with increased methionine dependency in breast cancer cells. Oncogenic PIK3CA was sufficient to confer methionine dependency to mammary epithelial cells, partly by decreasing cystine uptake through the transcriptional and posttranslational inhibition of xCT. Manipulation of xCT activity altered the proliferation of breast cancer cells in methionine-deficient, homocysteine-containing media, suggesting that it functionally contributed to methionine dependency. We propose that concurrent with decreased cystine uptake through xCT, PIK3CA mutant cells use homocysteine through the transsulfuration pathway to synthesize cysteine. Consequently, less homocysteine is available to produce methionine, contributing to methionine dependency. These results indicate that oncogenic PIK3CA alters methionine and cysteine utilization, partly by inhibiting xCT to contribute to the methionine dependency phenotype in breast cancer cells.

Najm, F.J., et al. Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens. Nat Biotechnol (2017).Abstract

Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The "Big Papi" (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.

Stewart-Ornstein, J., Cheng, H.W.J. & Lahav, G. Conservation and Divergence of p53 Oscillation Dynamics across Species. Cell Syst 5, 4, 410-417.e4 (2017).Abstract

The tumor-suppressing transcription factor p53 is highly conserved at the protein level and plays a key role in the DNA damage response. One important aspect of p53 regulation is its dynamics in response to DNA damage, which include oscillations. Here, we observe that, while the qualitative oscillatory nature of p53 dynamics is conserved across cell lines derived from human, monkey, dog, mouse, and rat, the oscillation period is variable. Specifically, rodent cells exhibit rapid p53 oscillations, whereas dog, monkey, and human cells show slower oscillations. Computational modeling and experiments identify stronger negative feedback between p53 and MDM2 as the driver of faster oscillations in rodents, suggesting that the period of oscillation is a network-level property. In total, our study shows that despite highly conserved signaling, the quantitative features of p53 oscillations can diverge across evolution. We caution that strong amino acid conservation of proteins and transcriptional network similarity do not necessarily imply conservation of time dynamics.

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