Using an ORF kinome screen in MCF-7 cells treated with the CDK4/6 inhibitor ribociclib plus fulvestrant, we identified FGFR1 as a mechanism of drug resistance. FGFR1-amplified/ER+ breast cancer cells and MCF-7 cells transduced with FGFR1 were resistant to fulvestrant ± ribociclib or palbociclib. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Addition of the FGFR TKI erdafitinib to palbociclib/fulvestrant induced complete responses of FGFR1-amplified/ER+ patient-derived-xenografts. Next generation sequencing of circulating tumor DNA (ctDNA) in 34 patients after progression on CDK4/6 inhibitors identified FGFR1/2 amplification or activating mutations in 14/34 (41%) post-progression specimens. Finally, ctDNA from patients enrolled in MONALEESA-2, the registration trial of ribociclib, showed that patients with FGFR1 amplification exhibited a shorter progression-free survival compared to patients with wild type FGFR1. Thus, we propose breast cancers with FGFR pathway alterations should be considered for trials using combinations of ER, CDK4/6 and FGFR antagonists.

The PI3K pathway is mutated and aberrantly activated in many cancers and plays a central role in tumor cell proliferation and survival, making it a rational therapeutic target. Until recently, however, results from clinical trials with PI3K inhibitors in solid tumors have been largely disappointing. Here, we describe several factors that have limited the success of these agents, including the weak driver oncogenic activity of mutant PI3K, suboptimal patient selection in trials, drug-related toxicities, feedback upregulation of compensatory mechanisms when PI3K is blocked, increased insulin production upon PI3Kα inhibition, lack of mutant-specific inhibitors, and a relative scarcity of studies using combinations with PI3K antagonists. We also suggest strategies to improve the impact of these agents in solid tumors. Despite these challenges, we are optimistic that isoform-specific PI3K inhibitors, particularly in combination with other agents, may be valuable in treating appropriately selected patients with PI3K-dependent tumors.Significance: Despite the modest clinical activity of PI3K inhibitors in solid tumors, there is an increasing understanding of the factors that may have limited their success. Strategies to ameliorate drug-related toxicities, use of rational combinations with PI3K antagonists, development of mutant-selective PI3K inhibitors, and better patient selection should improve the success of these targeted agents against solid tumors.

We present an oblique plane microscope (OPM) that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of lattice light-sheet microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.

Human epidermal growth factor receptor 2 ( HER2 ; ERBB2 ) amplification and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha ( PIK3CA ) mutations often co-occur in breast cancer. Aberrant activation of the phosphatidylinositol 3-kinase (PI3K) pathway has been shown to correlate with a diminished response to HER2-directed therapies. We generated a mouse model of HER2-overexpressing (HER2 ⁺ ), PIK3CA ^H1047R -mutant breast cancer. Mice expressing both human HER2 and mutant PIK3CA in the mammary epithelium developed tumors with shorter latencies compared with mice expressing either oncogene alone. HER2 and mutant PIK3CA also cooperated to promote lung metastases. By microarray analysis, HER2-driven tumors clustered with luminal breast cancers, whereas mutant PIK3CA tumors were associated with claudin-low breast cancers. PIK3CA and HER2 ⁺ / PIK3CA tumors expressed elevated transcripts encoding markers of epithelial-to-mesenchymal transition and stem cells. Cells from HER2 ⁺ / PIK3CA tumors more efficiently formed mammospheres and lung metastases. Finally, HER2 ⁺ / PIK3CA tumors were resistant to trastuzumab alone and in combination with lapatinib or pertuzumab. Both drug resistance and enhanced mammosphere formation were reversed by treatment with a PI3K inhibitor. In sum, PIK3CA ^H1047R accelerates HER2-mediated breast epithelial transformation and metastatic progression, alters the intrinsic phenotype of HER2-overexpressing cancers, and generates resistance to approved combinations of anti-HER2 therapies.

Detection of minor, genetically distinct subpopulations within tumors is a key challenge in cancer genomics. Here we report STAR-FISH (specific-to-allele PCR–FISH), a novel method for the combined detection of single-nucleotide and copy number alterations in single cells in intact archived tissues. Using this method, we assessed the clinical impact of changes in the frequency and topology of PIK3CA mutation and HER2 (ERBB2) amplification within HER2-positive breast cancer during neoadjuvant therapy. We found that these two genetic events are not always present in the same cells. Chemotherapy selects for PIK3CA-mutant cells, a minor subpopulation in nearly all treatment-naive samples, and modulates genetic diversity within tumors. Treatment-associated changes in the spatial distribution of cellular genetic diversity correlated with poor long-term outcome following adjuvant therapy with trastuzumab. Our findings support the use of in situ single cell–based methods in cancer genomics and imply that chemotherapy before HER2-targeted therapy may promote treatment resistance.