Weighing the Evidence: Variant Classification and Interpretation in Precision Oncology, US Food and Drug Administration Public Workshop-Workshop Proceedings.

PURPOSE: Next-generation sequencing (NGS) oncology panels are becoming integral in hospital and academic settings to guide patient treatment and enrollment in clinical trials. Although NGS technologies have revolutionized decision-making for cancer therapeutics, physicians may face many challenges in parsing and prioritizing NGS-based test results to determine the best course of treatment for individual patients. On January 29, 2018, the US Food and Drug Administration held a public workshop entitled, "Weighing the Evidence: Variant Classification and Interpretation in Precision Oncology." Here, we discuss the presentations and discussion highlights across the four sessions of the workshop. METHODS: The goal of the public workshop was to engage stakeholders and solicit input from experts in precision oncology to discuss the integration of complex NGS data into patient management and regulatory innovation within the precision oncology community. The US Food and Drug Administration gathered representatives from academia, industry, patient advocacy, government, and professional organizations for a series of presentations followed by panel discussions. After the workshop, the transcript and speaker presentation slides were reviewed and summarized for manuscript preparation. RESULTS: Speakers and panelists provided diverse perspectives on the integration of NGS technology into patient care for oncology and on the complexities that surround data interpretation and sharing. Discussions highlighted the challenges with standardization for variant classification while expressing the utility of consensus recommendations among stakeholders in oncology for driving innovation in the era of precision medicine. CONCLUSION: As precision medicine advances, clear communication within the field of precision oncology will be key to creating an environment that facilitates the generation and sharing of data that have value to patients.

Engineering allosteric regulation in protein kinases.

Phosphoregulation, in which the addition of a negatively charged phosphate group modulates protein activity, enables dynamic cellular responses. To understand how new phosphoregulation might be acquired, we mutationally scanned the surface of a prototypical yeast kinase (Kss1) to identify potential regulatory sites. The data revealed a set of spatially distributed "hotspots" that might have coevolved with the active site and preferentially modulated kinase activity. By engineering simple consensus phosphorylation sites at these hotspots, we rewired cell signaling in yeast. Using the same approach with a homolog yeast mitogen-activated protein kinase, Hog1, we introduced new phosphoregulation that modified its localization and signaling dynamics. Beyond revealing potential use in synthetic biology, our findings suggest that the identified hotspots contribute to the diversity of natural allosteric regulatory mechanisms in the eukaryotic kinome and, given that some are mutated in cancers, understanding these hotspots may have clinical relevance to human disease.

Hierarchical Organization Endows the Kinase Domain with Regulatory Plasticity.

The functional diversity of kinases enables specificity in cellular signal transduction. Yet how more than 500 members of the human kinome specifically receive regulatory inputs and convey information to appropriate substrates-all while using the common signaling output of phosphorylation-remains enigmatic. Here, we perform statistical co-evolution analysis, mutational scanning, and quantitative live-cell assays to reveal a hierarchical organization of the kinase domain that facilitates the orthogonal evolution of regulatory inputs and substrate outputs while maintaining catalytic function. We find that three quasi-independent "sectors"-groups of evolutionarily coupled residues-represent functional units in the kinase domain that encode for catalytic activity, substrate specificity, and regulation. Sector positions impact both disease and pharmacology: the catalytic sector is significantly enriched for somatic cancer mutations, and residues in the regulatory sector interact with allosteric kinase inhibitors. We propose that this functional architecture endows the kinase domain with inherent regulatory plasticity.

SMARCE1 is required for the invasive progression of in situ cancers.

Advances in mammography have sparked an exponential increase in the detection of early-stage breast lesions, most commonly ductal carcinoma in situ (DCIS). More than 50% of DCIS lesions are benign and will remain indolent, never progressing to invasive cancers. However, the factors that promote DCIS invasion remain poorly understood. Here, we show that SMARCE1 is required for the invasive progression of DCIS and other early-stage tumors. We show that SMARCE1 drives invasion by regulating the expression of secreted proteases that degrade basement membrane, an ECM barrier surrounding all epithelial tissues. In functional studies, SMARCE1 promotes invasion of in situ cancers growing within primary human mammary tissues and is also required for metastasis in vivo. Mechanistically, SMARCE1 drives invasion by forming a SWI/SNF-independent complex with the transcription factor ILF3. In patients diagnosed with early-stage cancers, SMARCE1 expression is a strong predictor of eventual relapse and metastasis. Collectively, these findings establish SMARCE1 as a key driver of invasive progression in early-stage tumors.

Defining the Essential Function of Yeast Hsf1 Reveals a Compact Transcriptional Program for Maintaining Eukaryotic Proteostasis.

Despite its eponymous association with the heat shock response, yeast heat shock factor 1 (Hsf1) is essential even at low temperatures. Here we show that engineered nuclear export of Hsf1 results in cytotoxicity associated with massive protein aggregation. Genome-wide analysis revealed that Hsf1 nuclear export immediately decreased basal transcription and mRNA expression of 18 genes, which predominately encode chaperones. Strikingly, rescuing basal expression of Hsp70 and Hsp90 chaperones enabled robust cell growth in the complete absence of Hsf1. With the exception of chaperone gene induction, the vast majority of the heat shock response was Hsf1 independent. By comparative analysis of mammalian cell lines, we found that only heat shock-induced but not basal expression of chaperones is dependent on the mammalian Hsf1 homolog (HSF1). Our work reveals that yeast chaperone gene expression is an essential housekeeping mechanism and provides a roadmap for defining the function of HSF1 as a driver of oncogenesis.

GSK-3ß Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility.

Glycogen synthase kinase 3 (GSK-3) has been linked to regulation of kinesin-dependent axonal transport in squid and flies, and to indirect regulation of cytoplasmic dynein. We have now found evidence for direct regulation of dynein by mammalian GSK-3ß in both neurons and non-neuronal cells. GSK-3ß coprecipitates with and phosphorylates mammalian dynein. Phosphorylation of dynein intermediate chain (IC) reduces its interaction with Ndel1, a protein that contributes to dynein force generation. Two conserved residues, S87/T88 in IC-1B and S88/T89 in IC-2C, have been identified as GSK-3 targets by both mass spectrometry and site-directed mutagenesis. These sites are within an Ndel1-binding domain, and mutation of both sites alters the interaction of IC's with Ndel1. Dynein motility is stimulated by (i) pharmacological and genetic inhibition of GSK-3ß, (ii) an insulin-sensitizing agent (rosiglitazone) and (iii) manipulating an insulin response pathway that leads to GSK-3ß inactivation. Thus, our study connects a well-characterized insulin-signaling pathway directly to dynein stimulation via GSK-3 inhibition.

A Cdk5-dependent switch regulates Lis1/Ndel1/dynein-driven organelle transport in adult axons.

Lissencephaly is a human developmental brain abnormality caused by LIS1 haploinsufficiency. This disorder is in large part attributed to altered mitosis and migration in the developing brain. LIS1 and an interacting protein, NDEL1, bind to cytoplasmic dynein, a microtubule motor protein. While the tripartite complex is clearly important for developmental events, we are intrigued by the fact that Lis1 and Ndel1 expression remain high in the adult mouse nervous system. Dynein plays a crucial role in retrograde axonal transport, a process that is used by mature neurons. Here, we monitored acidic organelles moving in axons of adult rat sensory neurons to determine whether Lis1 and Ndel1 contribute to axonal transport. Lis1 RNAi significantly reduced axon transport of these organelles. Ndel1 RNAi had little impact, but combined Lis1 and Ndel1 RNAi caused a more severe phenotype than Lis1 RNAi alone, essentially shutting down transport. Lis1 overexpression stimulated retrograde transport, while a Lis1 dynein-binding mutant severely disrupted transport. Overexpression of Ndel1 or a Lis1 Ndel1-binding mutant only mildly perturbed transport. However, expressing a mutant Ndel1 lacking key phosphorylation sites shut down transport completely, as did a dominant-negative Cdk5 construct. We propose that, in axons, unphosphorylated Ndel1 inhibits the capacity of dynein to transport acidic organelles. Phosphorylation of Ndel1 by Cdk5 not only reduces this inhibition but also allows Lis1 to further stimulate the cargo transport capacity of dynein. Our data raise the possibility that defects in a Lis1/Ndel1 regulatory switch could contribute to neurodegenerative diseases linked to axonal pathology in adults.