Introduction: Modern targeted cancer therapies are carefully crafted small molecules. These exquisite technologies exhibit an astonishing diversity of failure modes (drug resistance mechanisms) in the clinic. This diversity is surprising because back of the envelope calculations and classic modeling results in evolutionary dynamics suggest that the diversity in the modes of clinical drug resistance should be considerably smaller than what is observed. These same calculations suggest that known microenvironmental resistance mechanisms should not be able to compete for outgrowth with genetic resistance within a tumor, and yet evidence of microenvironmental resistance is often observed in the clinic. Quantitatively understanding the underlying biological mechanisms of failure mode diversity may improve the next generation of targeted anticancer therapies. It also provides insights into how intratumoral heterogeneity might shape interpatient diversity during clinical relapse. Materials and Methods: We employed spatial agent-based models to explore regimes where spatial constraints enable microenvironmental resistance to significantly compete with genetically resistant subclones. In order to parameterize a model of microenvironmental resistance, BT20 cells were cultured in the presence and absence of fibroblasts from 16 different tissues. The degree of resistance conferred by cancer associated fibroblasts (CAFs) in the tumor microenvironment was quantified by treating mono- and co-cultures with letrozole and then measuring the death rates. Results and Discussion: Our simulations indicate that, even when a mutation is more drug resistant, its outgrowth can be delayed by abundant, low magnitude microenvironmental resistance across large regions of a tumor. These observations hold for different modes of microenvironmental resistance, including juxtacrine signaling, soluble secreted factors, and remodeled ECM. This result helps to explain the remarkable diversity of resistance mechanisms observed in solid tumors, which subverts the presumption that the failure mode that causes the quantitatively fastest growth in the presence of drug should occur most often in the clinic. Conclusion: Our model results demonstrate that spatial effects can interact with low magnitude of resistance microenvironmental effects to successfully compete against genetic resistance that is orders of magnitude larger. Clinical outcomes of solid tumors are intrinsically connected to their spatial structure, and the tractability of spatial agent-based models like the ones presented here enable us to understand this relationship more completely.

The development of new drug regimens that allow rapid, sterilizing treatment of tuberculosis has been limited by the complexity and time required for genetic manipulations in Mycobacterium tuberculosis. CRISPR interference (CRISPRi) promises to be a robust, easily engineered and scalable platform for regulated gene silencing. However, in M. tuberculosis, the existing Streptococcus pyogenes Cas9-based CRISPRi system is of limited utility because of relatively poor knockdown efficiency and proteotoxicity. To address these limitations, we screened eleven diverse Cas9 orthologues and identified four that are broadly functional for targeted gene knockdown in mycobacteria. The most efficacious of these proteins, the CRISPR1 Cas9 from Streptococcus thermophilus (dCas9Sth1), typically achieves 20- to 100-fold knockdown of endogenous gene expression with minimal proteotoxicity. In contrast to other CRISPRi systems, dCas9Sth1-mediated gene knockdown is robust when targeted far from the transcriptional start site, thereby allowing high-resolution dissection of gene function in the context of bacterial operons. We demonstrate the utility of this system by addressing persistent controversies regarding drug synergies in the mycobacterial folate biosynthesis pathway. We anticipate that the dCas9Sth1 CRISPRi system will have broad utility for functional genomics, genetic interaction mapping and drug-target profiling in M. tuberculosis.