In vivo E2F reporting reveals efficacious schedules of MEK1/2–CDK4/6 targeting and mTOR–s6 resistance mechanisms

Targeting cyclin-dependent kinases 4/6 (CDK4/6) represents a therapeutic option in combination with BRAF inhibitor and/or MEK inhibitor (MEKi) in melanoma; however, continuous dosing elicits toxicities in patients. Using quantitative and temporal in vivo reporting, we show that continuous MEKi with intermittent CDK4/6 inhibitor (CDK4/6i) led to more complete tumor responses versus other combination schedules. Nevertheless, some tumors acquired resistance that was associated with enhanced phosphorylation of ribosomal S6 protein. These data were supported by phospho-S6 staining of melanoma biopsies from patients treated with CDK4/6i plus targeted inhibitors. Enhanced phospho-S6 in resistant tumors provided a therapeutic window for the mTORC1/2 inhibitor AZD2014. Mechanistically, upregulation or mutation of NRAS was associated with resistance in in vivo models and patient samples, respectively, and mutant NRAS was sufficient to enhance resistance. This study utilizes an in vivo reporter model to optimize schedules and supports targeting mTORC1/2 to overcome MEKi plus CDK4/6i resistance. SIGnIFICAnCE: Mutant BRAF and NRAS melanomas acquire resistance to combined MEK and CDK4/6 inhibition via upregulation of mTOR pathway signaling. This resistance mechanism provides the preclinical basis to utilize mTORC1/2 inhibitors to improve MEKi plus CDK4/6i drug regimens

A preexisting rare PIK3CA e545k subpopulation confers clinical resistance to MEK plus CDK4/6 inhibition in NRAS melanoma and is dependent on S6K1 signaling

Combined MEK and CDK4/6 inhibition (MEKi + CDK4i) has shown promising clinical outcomes in patients with NRAS- mutant melanoma. Here, we interrogated longitudinal biopsies from a patient who initially responded to MEKi + CDK4i therapy but subsequently developed resistance. Whole-exome sequencing and functional validation identified an acquired PIK3CA E545K mutation as conferring drug resistance. We demonstrate that PIK3CA E545K preexisted in a rare subpopulation that was missed by both clinical and research testing, but was revealed upon multiregion sampling due to PIK3CA E545K being nonuniformly distributed. This resistant population rapidly expanded after the initiation of MEKi + CDK4i therapy and persisted in all successive samples even after immune checkpoint therapy and distant metastasis. Functional studies identified activated S6K1 as both a key marker and specific therapeutic vulnerability downstream of PIK3CA E545K -induced resistance. These results demonstrate that difficult-to-detect preexisting resistance mutations may exist more often than previously appreciated and also posit S6K1 as a common downstream therapeutic nexus for the MAPK, CDK4/6, and PI3K pathways. SIGNIFICANCE: We report the first characterization of clinical acquired resistance to MEKi + CDK4i, identifying a rare preexisting PIK3CA E545K subpopulation that expands upon therapy and exhibits drug resistance. We suggest that single-region pretreatment biopsy is insufficient to detect rare, spatially segregated drug-resistant subclones. Inhibition of S6K1 is able to resensitize PIK3CA E545K -expressing NRAS-mutant melanoma cells to MEKi + CDK4i. © 2018 AAC

Treating Solar Model Uncertainties: A Consistent Statistical Analysis of Solar Neutrino Models and Data

We describe how to consistently incorporate solar model uncertainties, along with experimental errors and correlations, when analyzing solar neutrino data to derive confidence limits on parameter space for proposed solutions of the solar neutrino problem. Our work resolves ambiguities and inconsistencies in the previous literature. As an application of our methods we calculate the masses and mixing angles allowed by the current data for the proposed MSW solution using both Bayesian and frequentist methods, allowing purely for solar model flux variations, to compare with previous work. We consider the effects of including metal diffusion in the solar models and also discuss implications for future experiments.Comment: 29 pages (incl figs), latex, 6 figures (appended as separate uuencoded file. To embed figures in text, uncomment 6 \epsfysize lines which appear before bibliography), CWRU-P5-94, CfPA-94-TTH-29, Fermilab-Pub-94/176-

Clinical Profiling of BCL-2 Family Members in the Setting of BRAF Inhibition Offers a Rationale for Targeting De Novo Resistance Using BH3 Mimetics

While response rates to BRAF inhibitiors (BRAFi) are high, disease progression emerges quickly. One strategy to delay the onset of resistance is to target anti-apoptotic proteins such as BCL-2, known to be associated with a poor prognosis. We analyzed BCL-2 family member expression levels of 34 samples from 17 patients collected before and 10 to 14 days after treatment initiation with either vemurafenib or dabrafenib/trametinib combination. The observed changes in mRNA and protein levels with BRAFi treatment led us to hypothesize that combining BRAFi with a BCL-2 inhibitor (the BH3-mimetic navitoclax) would improve outcome. We tested this hypothesis in cell lines and in mice. Pretreatment mRNA levels of BCL-2 negatively correlated with maximal tumor regression. Early increases in mRNA levels were seen in BIM, BCL-XL, BID and BCL2-W, as were decreases in MCL-1 and BCL2A. No significant changes were observed with BCL-2. Using reverse phase protein array (RPPA), significant increases in protein levels were found in BIM and BID. No changes in mRNA or protein correlated with response. Concurrent BRAF (PLX4720) and BCL2 (navitoclax) inhibition synergistically reduced viability in BRAF mutant cell lines and correlated with down-modulation of MCL-1 and BIM induction after PLX4720 treatment. In xenograft models, navitoclax enhanced the efficacy of PLX4720. The combination of a selective BRAF inhibitor with a BH3-mimetic promises to be an important therapeutic strategy capable of enhancing the clinical efficacy of BRAF inhibition in many patients that might otherwise succumb quickly to de novo resistance. Trial Registrations: ClinicalTrials.gov NCT01006980; ClinicalTrials.gov NCT01107418; ClinicalTrials.gov NCT01264380; ClinicalTrials.gov NCT01248936; ClinicalTrials.gov NCT00949702; ClinicalTrials.gov NCT0107217

<html>Efficacy of the combination of MEK and CDK4/6 inhibitors <i>in vitro</i> and <i>in vivo</i> in KRAS mutant colorectal cancer models</html>

Though the efficacy of MEK inhibitors is being investigated in KRAS-mutant colorectal cancers (CRC), early clinical trials of MEK inhibitor monotherapy did not reveal significant antitumor activity. Resistance to MEK inhibitor monotherapy developed through a variety of mechanisms converging in ERK reactivation. Since ERK increases cyclin D expression and increases entry into the cell cycle, we hypothesized that the combination of MEK inhibitors and CDK4/6 inhibitors would have synergistic antitumor activity and cause tumor regression in vivo

Deep sequencing with longitudinal sampling of a VRC01-like-antibody response in a chronically infected individual

Background: VRC01-like antibodies use heavy chain mimicry of the CD4-receptor to achieve effective neutralization of HIV-1. The VRC01-like antibodies that have been observed in a number of HIV-1-infected individuals (i) display extensive somatic changes (70-100 nucleotide changes in VH-gene), (ii) can be detected only after several years of infection, (iii) derive from VH1-2, and (iv) are compatible with several different heavy J chains and different light chains. Methods: To understand the persistence, evolution, and lineage of VRC01-like antibodies, we sampled PBMCs from donor 45, the source of VRC01 and VRC03 antibodies, at approximately yearly intervals over a 15-year period, and performed deep sequencing on the heavy and light chain variable portions of expressed antibodies. Anti-idiotypic antibodies were used to correlate mRNA levels of antibodies identified by the deep sequencing with expressed levels of these antibodies in serum. Results: High expression levels of VRC01-like antibody sequences persisted over the entire 15-year period. Multiple lineages of VRC01-like antibodies were detected at each time point, and some of these, in particular the lineages that include VRC01 and VRC03, persisted over multiple time points, and displayed extensive branching in their evolution. Conclusion: Deep sequencing provides a means to define the genetic record of the lineage and maturation of antibodies effective at neutralizing HIV-1. Precise definition of the natural ontogeny of broadly neutralizing antibodies may be essential in defining appropriate strategies to elicit such antibodies in vaccine settings

Ligand-Receptor Interactions

The formation and dissociation of specific noncovalent interactions between a variety of macromolecules play a crucial role in the function of biological systems. During the last few years, three main lines of research led to a dramatic improvement of our understanding of these important phenomena. First, combination of genetic engineering and X ray cristallography made available a simultaneous knowledg of the precise structure and affinity of series or related ligand-receptor systems differing by a few well-defined atoms. Second, improvement of computer power and simulation techniques allowed extended exploration of the interaction of realistic macromolecules. Third, simultaneous development of a variety of techniques based on atomic force microscopy, hydrodynamic flow, biomembrane probes, optical tweezers, magnetic fields or flexible transducers yielded direct experimental information of the behavior of single ligand receptor bonds. At the same time, investigation of well defined cellular models raised the interest of biologists to the kinetic and mechanical properties of cell membrane receptors. The aim of this review is to give a description of these advances that benefitted from a largely multidisciplinar approach