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Background Combined targeting of CDK4/6 and ER is now the standard of care for patients with advanced ER+/HER2− breast cancer. However, acquired resistance to these therapies frequently leads to disease progression. As such, it is critical to identify the mechanisms by which resistance to CDK4/6-based therapies is acquired and also identify therapeutic strategies to overcome resistance. Methods In this study, we developed and characterized multiple in vitro and in vivo models of acquired resistance to CDK4/6-based therapies. Resistant models were screened by reverse phase protein array (RPPA) for cell signaling changes that are activated in resistance. Results We show that either a direct loss of Rb or loss of dependence on Rb signaling confers cross-resistance to inhibitors of CDK4/6, while PI3K/mTOR signaling remains activated. Treatment with the p110α-selective PI3K inhibitor, alpelisib (BYL719), completely blocked the progression of acquired CDK4/6 inhibitor-resistant xenografts in the absence of continued CDK4/6 inhibitor treatment in models of both PIK3CA mutant and wild-type ER+/HER2− breast cancer. Triple combination therapy against PI3K:CDK4/6:ER prevented and/or delayed the onset of resistance in treatment-naive ER+/HER2− breast cancer models. Conclusions These data support the clinical investigation of p110α-selective inhibitors of PI3K, such as alpelisib, in patients with ER+/HER2− breast cancer who have progressed on CDK4/6:ER-based therapies. Our data also support the investigation of PI3K:CDK4/6:ER triple combination therapy to prevent the onset of resistance to the combination of endocrine therapy plus CDK4/6 inhibition.

The bis-tetrahydroisoquinoline (bis-THIQ) natural products have been studied intensively over the past four decades for their exceptionally potent anticancer activity, in addition to strong Gram-positive and Gram-negative antibiotic character. Synthetic strategies toward these complex polycyclic compounds have relied heavily on electrophilic aromatic chemistry, such as the Pictet–Spengler reaction, that mimics their biosynthetic pathways. Herein, we report an approach to two bis-THIQ natural products, jorunnamycin A and jorumycin, that instead harnesses the power of modern transition-metal catalysis for the three major bond-forming events and proceeds with high efficiency (15 and 16 steps, respectively). By breaking from biomimicry, this strategy allows for the preparation of a more diverse set of nonnatural analogs.

Everolimus (RAD001, Afinitor ® ) is an oral, selective mTOR inhibitor recently approved by the US-FDA in combination with exemestane for treatment of hormone receptor positive advanced breast cancer. To date, no molecular predictors of response to everolimus in breast cancer have been identified. We hypothesized predictive markers could be identified using preclinical models. Using a molecularly characterized panel of human breast cancer and immortalized breast epithelial cell lines, we determined sensitivity to everolimus alone or in combination with ER− or HER2− targeted therapy. Gene expression microarrays and comparative genomic hybridization were performed on the cell lines to identify predictors of response to everolimus. Among 13 everolimus-sensitive cell lines, 10/13(77 %) were luminal, while in 26 resistant cell lines, 16/26(62 %) were non-luminal, and 10/26(38 %) were luminal. Only 3/24 non-luminal lines were sensitive, two of which were HER2+. Everolimus enhanced the anti-proliferative effect of both tamoxifen (TAM) and fulvestrant (FUL) in ER+ breast cancer cell lines, as well as trastuzumab in HER2+ cell lines. Everolimus + FUL but not everolimus + TAM reversed acquired resistance to TAM. Everolimus inhibited mTOR in tested cell lines by decreasing S6 phosphorylation, mediating its anti-proliferative effect by G0/G1 cell cycle arrest and induction of apoptosis. Chromosomal amplifications of AURKA ( p value = 0.04) and HER2 ( p value = 0.03) were each associated with increased sensitivity to everolimus. Transcript expression microarrays identified GSK3A, PIK3R3, KLF8 , and MAPK10 among the genes overexpressed in sensitive luminal lines, while PGP, RPL38, GPT , and GFAP were among the genes overexpressed in resistant luminal cell lines. These preclinical in vitro data provide further support for continued clinical development of everolimus in luminal (ER+ or HER2+) breast cancer in combination with targeted therapies. We identified several potential molecular markers associated with response to everolimus that will require validation in clinical material.

Aurora kinases play important roles in cell division and are frequently overexpressed in human cancer. AMG 900 is a novel pan-Aurora kinase inhibitor currently being tested in Phase I clinical trials. We aimed to evaluate the in vitro activity of AMG 900 in a panel of 44 human breast cancer and immortalized cell lines and identify predictors of response. AMG 900 inhibited proliferation at low nanomolar concentrations in all cell lines tested. Response was further classified based on the induction of lethality. 25 cell lines were classified as highly sensitive (lethality at 10 nM of AMG 900 >10 %), 19 cell lines as less sensitive to AMG 900 (lethality at 10 nM of AMG 900 <10 %). Traditional molecular subtypes of breast cancer did not predict for this differential response. There was a weak association between AURKA amplification and response to AMG 900 (response ratio = 2.53, p  = 0.09). mRNA expression levels of AURKA , AURKB , and AURKC and baseline protein levels of Aurora kinases A and B did not significantly associate with response. Cell lines with TP53 loss of function mutations (RR = 1.86, p  = 0.004) and low baseline p21 protein levels (RR = 2.28, p  = 0.0004) were far more likely to be classified as highly sensitive to AMG 900. AMG 900 induced p53 and p21 protein expression in cell lines with wt TP53 . AMG 900 caused the accumulation of cells with >4 N DNA content in a majority of cell lines independently of sensitivity and p53 status. AMG 900 induced more pronounced apoptosis in highly sensitive p53-dysfunctional cell lines. We have found that AMG 900 is highly active in breast cancer cell lines and that TP53 loss of function mutations as well as low baseline expression of p21 protein predict strongly for increased sensitivity to this compound in vitro.

Palbociclib is a highly specific small molecule inhibitor of cyclin-dependent kinases 4 and 6 currently in clinical development by Pfizer. It is designed to inhibit the cell cycle at the G1/S transition via preventing the phosphorylation of Rb by the Cyclin D/CDK4/6 enzyme complex. Our lab previously identified the estrogen receptor positive subpopulation of breast cancer patients as distinctively likely to benefit from treatment with palbociclib. This observation spurred the initiation of a Phase II clinical trial in this patient population where remarkable efficacy was observed. Given this translational success, we wished to investigate the molecular determinants of response to palbociclib in several additional cancer types. To this end, we assayed the in vitro sensitivity to palbociclib across a panel of 416 cancer cell lines derived from 12 distinct cancer types. We observed highly differential response to treatment both within and between cancer types. IC50s (the concentration of palbociclib required to inhibit fifty percent of population doublings) ranged from the low nanomolar range to above the highest dose tested (1µM). This response distribution was much broader than was observed in similar screens performed by the Broad and Sanger institutes, where the vast majority of cell lines assayed were listed as having IC50s above 1uM. Our ability to identify a higher proportion of palbociclib -sensitive cell lines can likely be credited to methodological innovations aimed at optimizing our screening protocol for the detection of longer-term cytostatic effects, as opposed to shorter-term cytotoxic effects of treatment. The generation of this highly differential response dataset allowed for a unique opportunity to explore the possible genetic mechanisms underlying differential sensitivity to treatment with palbociclib in vitro. The other dataset interrogated for genotype-response associations was a copy number alteration dataset derived from comparative genomic hybridization arrays. This dataset was organized by gene and also restricted by various criteria to enrich for amplifications or deletions of genes likely to be causally involved in carcinogenesis. From this dataset we identified three chromosomal regions (17q12-21, 11q13, 1q32) where amplification was associated with sensitivity to palbociclib. Two amplified regions (19q13, 8q13) were found to be associated with resistance. Homozygous deletions of the 13q14 chromosomal region were found to be strongly associated with resistance to palbociclib. Following these extensive post-hoc analyses, we pruned our original set of fourteen biomarkers down to the eight most likely to play a causal role in determining sensitivity or resistance to treatment with palbociclib. The final set of candidate sensitivity biomarkers included: loss-of-function point mutations in CDH1, loss-of-function point mutations in TOPBP1, chromosomal amplification of 17q12-21 (ERBB2) and chromosomal amplification of 11q13 (CCND1). The final set of candidate resistance biomarkers included: activating point mutations in SMO, chromosomal amplification of CCNE1, loss-of-function point mutations in RB1 and chromosomal deletion of 13q14 (RB1). This final set of eight candidate biomarkers was analyzed by strata representing each of the 12 cancer types in our cell line panel. The four candidate sensitivity biomarkers were sufficiently frequent and associated with sensitivity in 6 of the 12 cancer types in our panel. These were the breast, colon, head/neck, lung, ovarian and upper gastrointestinal strata. The four candidate resistance biomarkers were sufficiently frequent and associated with resistance in 6 of the 12 cancer types in our panel. These were the breast, colon, kidney, lung, ovarian and upper gastrointestinal strata. (Abstract shortened by UMI.)

In the emergency department, troponin assays are commonly used and essential in the evaluation of chest pain and diagnosis of acute coronary syndrome. This study was designed to assess the potential impact of implementing point-of-care troponin testing by comparing the time to point-of-care laboratory result and time to conventional laboratory result. The study enrolled 60 ED patients deemed to need a troponin test in the evaluation of low-risk chest pain (HEART score <4 based on history, electrocardiogram, age, risk factors). Point-of-care troponin testing was performed with the same blood sample obtained for a conventional troponin assay. If the provider determined that the patient required 2 troponin tests, the second laboratory draw was used in the data collection. This was to correlate the time of laboratory result to time of disposition. Of the 60 subjects enrolled, 2 subjects were excluded because of user errors with the point-of-care testing equipment and 2 others for not meeting inclusion criteria on later review. The median times for the point-of-care troponin and conventional troponin assays were 11:00 minutes (interquartile range 10:00-15:30) and 40:00 minutes (interquartile range 31:30-52:30), respectively; P < 0.001. There were 3 extreme outliers from the conventional troponin assay that significantly skewed the distribution of the mean, making the median the more accurate assessment of the central tendency. Point-of-care troponin testing provided results in a median time 29 minutes quicker than the conventional troponin assay. This result is statistically significant and has the potential to greatly improve time to disposition in all patients with chest pain requiring a troponin assay.

Inactivation of disease alleles by allele-specific editing is a promising approach to treat dominant-negative genetic disorders, provided the causative gene is haplo-sufficient. We previously edited a dominant missense mutation with inactivating frameshifts and rescued disease-relevant phenotypes in induced pluripotent stem cell (iPSC)-derived motor neurons. However, a multitude of different missense mutations cause disease. Here, we addressed this challenge by targeting common single-nucleotide polymorphisms in cis with disease mutations for gene excision. We validated this haplotype editing approach for two different missense mutations and demonstrated its therapeutic potential in iPSC-motor neurons. Surprisingly, our analysis revealed that gene inversion, a frequent byproduct of excision editing, failed to reliably disrupt mutant allele expression. We deployed alternative strategies and novel molecular assays to increase therapeutic editing outcomes while maintaining specificity for the mutant allele. Finally, population genetics analysis demonstrated the power of haplotype editing to enable therapeutic development for the greatest number of patients. Our data serve as an important case study for many dominant genetic disorders amenable to this approach.