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Background: Autophagy inhibition is a potential treatment for central nervous system (CNS) tumors. Autophagy, a heavily regulated process by which cellular waste is transferred to lysosomes for degradation and processing, is an integral part of tumor cell survival under stressful conditions including nutrient deprivation and chemotherapy. While the efficacy of autophagy inhibition has been demonstrated in CNS tumors with BRAF^(V600e) mutations, it has yet to be explored in other CNS tumor types with MAPK pathway dysregulation including NF1-mutated tumors. Many tumors associated with the NF1 phenotype can be difficult to treat surgically thus development of further pharmacologic interventions is necessary. Methods: A CRISPR/Cas9 mediated NF1 KO was derived from human immortalized Schwann cells and utilized as a tumor model. Autophagy inhibition was achieved pharmacologically by chloroquine (CQ) and genetically via shRNAi of ATG5 and ATG7. Trametinib was used for MEK inhibition. Cell growth and viability were determined by Incucyte, Cell Titer-Glo luminescent assay, and colony-formation assays. Protein expression was measured by western blot. Results: We demonstrate increased autophagic activity in NF1 KO cell as compared to control lines both at baseline and in response to cellular stress. Furthermore, we describe that NF1 KO cells exhibit increased sensitivity to CQ alone, CQ in combination with trametinib, and shRNAi-mediated autophagy inhibition in combination with trametinib. Conclusion: Here, we describe increased autophagic dependence of NF1 mutated tumors and demonstrate increased tumor sensitivity to autophagy inhibition both alone and in combination with MEK inhibition. These findings indicate that autophagy inhibition via CQ may be an effective adjunctive treatment for NF1 mutated tumors and suggests that diverse CNS tumor types with MAPK pathway dysregulation are susceptible to autophagy inhibition. Clinical investigation of combined MEK and autophagy inhibition has the potential to improve outcomes for NF1 patients with CNS tumors.

Application of synthetic biology is limited by the capacity of cells to faithfully execute burdensome engineered functions in the face of Darwinian evolution. Division of labor, both metabolic and reproductive, are underutilized in confronting this barrier. To address this, we developed a serine-integrase based differentiation circuit that allows control of the population composition through tuning of the differentiation rate and number of cell divisions differentiated cells can undergo. We applied this system to T7 RNAP-driven expression of a fluorescent protein, and demonstrate both increased duration of circuit function and total production for high burden expression. While T7 expression systems are typically used for high-level short-term expression, this system enables longer duration production, and could be readily applied to burdensome or toxic products not readily produced in bacteria.

We recently developed 'autonomous hypermutation yeast surface display' (AHEAD), a technology that enables the rapid generation of potent and specific antibodies in yeast. AHEAD pairs yeast surface display with an error-prone orthogonal DNA replication system (OrthoRep) to continuously and rapidly mutate surface-displayed antibodies, thereby enabling enrichment for stronger binding variants through repeated rounds of cell growth and fluorescence activated cell sorting (FACS). AHEAD currently utilizes a standard galactose induction system to drive the selective display of antibodies on the yeast surface. However, achieving maximal display levels can require up to 48 hours of induction. Here, we report an updated version of the AHEAD platform that utilizes a synthetic β-estradiol induced gene expression system to regulate the surface display of antibodies and find that induction is notably faster in achieving surface display for both our AHEAD system as well as traditional yeast surface display from nuclear plasmids that do not hypermutate. The updated AHEAD platform was fully functional in repeated rounds of evolution to drive the rapid evolution of antibodies.

When nature maintains or evolves a gene's function over millions of years at scale, it produces a diversity of homologous sequences whose patterns of conservation and change contain rich structural, functional, and historical information about the gene. However, natural gene diversity likely excludes vast regions of functional sequence space and includes phylogenetic and evolutionary eccentricities, limiting what information we can extract. We introduce an accessible experimental approach for compressing long-term gene evolution to laboratory timescales, allowing for the direct observation of extensive adaptation and divergence followed by inference of structural, functional, and environmental constraints for any selectable gene. To enable this approach, we developed a new orthogonal DNA replication (OrthoRep) system that durably hypermutates chosen genes at a rate of >10 -4 substitutions per base in vivo . When OrthoRep was used to evolve a conditionally essential maladapted enzyme, we obtained thousands of unique multi-mutation sequences with many pairs >60 amino acids apart (>15% divergence), revealing known and new factors influencing enzyme adaptation. The fitness of evolved sequences was not predictable by advanced machine learning models trained on natural variation. We suggest that OrthoRep supports the prospective and systematic discovery of constraints shaping gene evolution, uncovering of new regions in fitness landscapes, and general applications in biomolecular engineering.