Faculty Bibliography

The PI3K pathway regulates proliferation, survival, and metabolism and is frequently activated across human cancers. A comprehensive elucidation of how this signaling pathway controls transcriptional and co-transcriptional processes could provide new insights into the key functions of PI3K signaling in cancer. Here, we undertook a transcriptomic approach to investigate genome-wide gene expression and transcription factor (TF) activity changes, as well as splicing and isoform usage dynamics, downstream of PI3K. These analyses uncovered widespread alternatively spliced (AS) isoforms linked to proliferation, metabolism, and splicing in PIK3CA mutant cells, which were reversed by inhibition of PI3Kα. Analysis of paired tumor biopsies from PIK3CA-mutated breast cancer patients undergoing treatment with PI3Kα inhibitors identified widespread splicing alterations that affect specific isoforms in common with the preclinical models, and these alterations, namely PTK2/FRNK and AFMID isoforms, were validated as functional drivers of cancer cell growth or migration. Mechanistically, isoform-specific splicing factors mediated PI3K-dependent RNA splicing. Treatment with splicing inhibitors rendered breast cancer cells more sensitive to the PI3Kα inhibitor alpelisib, resulting in greater growth inhibition than alpelisib alone. This study provides the first comprehensive analysis of widespread splicing alterations driven by oncogenic PI3K in breast cancer. The atlas of PI3K-mediated splicing programs establishes a key role for the PI3K pathway in regulating splicing, opening new avenues for exploiting PI3K signaling as a therapeutic vulnerability in breast cancer.

RASopathies are a group of genetic disorders that are caused by genes that affect the canonical Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Despite tremendous progress in understanding the molecular consequences of these genetic anomalies, little movement has been made in translating these findings to the clinic. This year, the seventh International RASopathies Symposium focused on expanding the research knowledge that we have gained over the years to enhance new discoveries in the field, ones that we hope can lead to effective therapeutic treatments. Indeed, for the first time, research efforts are finally being translated to the clinic, with compassionate use of Ras/MAPK pathway inhibitors for the treatment of RASopathies. This biannual meeting, organized by the RASopathies Network, brought together basic scientists, clinicians, clinician scientists, patients, advocates, and their families, as well as representatives from pharmaceutical companies and the National Institutes of Health. A history of RASopathy gene discovery, identification of new disease genes, and the latest research, both at the bench and in the clinic, were discussed.

RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. We have previously described a novel Cullin 3 RING E3 ubiquitin ligase complex formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of classical RAS GTPases. Here, we have analyzed the phenotypes of Lztr1 loss-of-function mutants in both fruit flies and mice and have demonstrated a biochemical preference for their RIT1 orthologs. Moreover, we show that Lztr1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of Rit1. Overall, our results indicate that, in model organisms, RIT1 orthologs are the preferred substrates of LZTR1.

Reciprocal interactions between endothelial cells (ECs) and adipocytes are fundamental to maintain white adipose tissue (WAT) homeostasis, as illustrated by the activation of angiogenesis upon WAT expansion, a process that is impaired in obesity. However, the molecular mechanisms underlying the crosstalk between ECs and adipocytes remain poorly understood. Here, we show that local production of polyamines in ECs stimulates adipocyte lipolysis and regulates WAT homeostasis in mice. We promote enhanced cell-autonomous angiogenesis by deleting Pten in the murine endothelium. Endothelial Pten loss leads to a WAT-selective phenotype, characterized by reduced body weight and adiposity in pathophysiological conditions. This phenotype stems from enhanced fatty acid β-oxidation in ECs concomitant with a paracrine lipolytic action on adipocytes, accounting for reduced adiposity. Combined analysis of murine models, isolated ECs and human specimens reveals that WAT lipolysis is mediated by mTORC1-dependent production of polyamines by ECs. Our results indicate that angiocrine metabolic signals are important for WAT homeostasis and organismal metabolism.

Understanding the molecular mechanisms that underlie different human pathologies is necessary to develop novel therapeutic strategies. An emerging mechanism of pathogenesis in many genetic disorders is the dysregulation of protein degradation, which leads to the accumulation of proteins that are responsible for the disease phenotype. Among the different cellular pathways that regulate active proteolysis, the Cullin RING E3 ligases represent an important group of sophisticated enzymatic complexes that mediate substrate ubiquitination through the interaction with specific adaptors. However, pathogenic variants in these adaptors affect the physiological ubiquitination of their substrates. This review discusses our current understanding of this emerging field.

The spindle assembly checkpoint (SAC) functions as a sensor of unattached kinetochores that delays mitotic progression into anaphase until proper chromosome segregation is guaranteed.¹

RAS proteins are molecular switches that interact with effector proteins when bound to guanosine triphosphate, stimulating downstream signaling in response to multiple stimuli. Although several canonical downstream effectors have been extensively studied and tested as potential targets for RAS-driven cancers, many of these remain poorly characterized. In this study, we undertook a biochemical and structural approach to further study the role of Sin1 as a RAS effector. Sin1 interacted predominantly with KRAS isoform 4A in cells through an atypical RAS-binding domain that we have characterized by X-ray crystallography. Despite the essential role of Sin1 in the assembly and activity of mTORC2, we find that the interaction with RAS is not required for these functions. Cells and mice expressing a mutant of Sin1 that is unable to bind RAS are proficient for activation and assembly of mTORC2. Our results suggest that Sin1 is a bona fide RAS effector that regulates downstream signaling in an mTORC2-independent manner.

PI3Kα inhibitors have shown clinical activity in PIK3CA-mutated estrogen receptor-positive (ER+) breast cancer patients. Using whole genome CRISPR/Cas9 sgRNA knockout screens, we identified and validated several negative regulators of mTORC1 whose loss confers resistance to PI3Kα inhibition. Among the top candidates were TSC1, TSC2, TBC1D7, AKT1S1, STK11, MARK2, PDE7A, DEPDC5, NPRL2, NPRL3, C12orf66, SZT2 and ITFG2. Loss of these genes invariably results in sustained mTOR signaling under pharmacological inhibition of the PI3K-AKT pathway. Moreover, resistance could be prevented or overcome by mTOR inhibition, confirming the causative role of sustained mTOR activity in limiting the sensitivity to PI3Kα inhibition. Cumulatively, genomic alterations affecting these genes are identified in about 15% of PIK3CA-mutated breast tumors and appear to be mutually exclusive. This study improves our understanding of the role of mTOR signaling restoration in leading to resistance to PI3Kα inhibition and proposes therapeutic strategies to prevent or revert this resistance.