Summary Advanced basal cell carcinomas (BCCs) frequently acquire resistance to Smoothened (SMO) inhibitors through unknown mechanisms. Here, we identify SMO mutations in 50% (22/44) of resistant BCCs and show that these mutations maintain Hedgehog signaling in the presence of SMO inhibitors. Alterations include four ligand binding pocket mutations defining sites of inhibitor binding and four variants confering constitutive activity and inhibitor resistance, illuminating pivotal residues that ensure receptor autoinhibition. In the presence of a SMO inhibitor, tumor cells containing either class of SMO mutants effectively outcompete cells containing the wild type SMO. Finally, we show that both classes of SMO variants respond to aPKC-ι/λ or GLI2 inhibitors that operate downstream of SMO, setting the stage for the clinical use of GLI antagonists.

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Nanomedicines (NMs) offer new solutions for cancer diagnosis and therapy. However, extension of progression-free interval and overall survival time achieved by Food and Drug Administrationapproved NMs remain modest. To develop next generation NMs to achieve superior anticancer activities, it is crucial to investigate and understand the correlation between the physicochemical properties of NMs (particle size in particular) and their interactions with biological systems to establish criteria for NM optimization. Here, we systematically evaluated the size-dependent biological profiles of three monodisperse drug-silica nanoconjugates (NCs; 20, 50, and 200 nm) through both experiments and mathematical modeling and aimed to identify the optimal size for the most effective anticancer drug delivery. Among the three NCs investigated, the 50-nm NC shows the highest tumor tissue retention integrated over time, which is the collective outcome of deep tumor tissue penetration and efficient cancer cell internalization as well as slow tumor clearance, and thus, the highest efficacy against both primary and metastatic tumors in vivo.O ver the last two to three decades, consensus has been reached that the size of anticancer nanomedicines (NMs) plays a pivotal role in determining their biodistribution, tumor penetration, cellular internalization, and clearance from blood plasma and tissues as well as excretion from body, and thus, it has significant impact on overall therapeutic efficacy against cancers (1-7). Although most clinically approved anticancer NMs have size ranging from 100 to 200 nm (8, 9), recent studies showed that anticancer NMs with smaller sizes exhibited enhanced performance in vivo, such as greater tissue penetration and enhanced tumor inhibition, particularly those with size around or smaller than 50 nm (5-7, 10-12). As such, there has been a major push recently in the field of anticancer NM to miniaturize nanoparticle (NP) size using novel chemistry and engineering design (13-17). One unanswered question, however, is whether additional miniaturization of NM size would be necessary and result in additional improved anticancer efficacy. Widely evaluated small molecular therapeutics (<1,500 Da and <2 nm) can traverse most tumor tissues freely (18). However, they diffuse away from tumor tissues rapidly and get cleared primarily into tumor blood capillaries, leading to minimal tumor accumulation (18). Macromolecules of relatively low molecular masses (<40,000 Da and <10 nm) were also shown to have low overall tumor retention because of both rapid permeation into and clearance from tumor tissues, behaving to some extent like small molecule drugs (18,19). In conjunction with the renal clearance threshold (<10-15 nm) (20, 21) and interstitial/lymphatic fenestration (<20 nm) (22) for NPs, it becomes essential to carefully and comprehensively evaluate the in vivo behavior and anticancer efficacy of NMs in the size range of 20-50 nm to determine the optimal size of NM for cancer therapy.In this study, we used monodisper...