Inferring tumor-specific cancer dependencies through integrating ex vivo drug response assays and drug-protein profiling

The development of cancer therapies may be improved by the discovery of tumor-specific molecular dependencies. The requisite tools include genetic and chemical perturbations, each with its strengths and limitations. Chemical perturbations can be readily applied to primary cancer samples at large scale, but mechanistic understanding of hits and further pharmaceutical development is often complicated by the fact that a chemical compound has affinities to multiple proteins. To computationally infer specific molecular dependencies of individual cancers from their ex vivo drug sensitivity profiles, we developed a mathematical model that deconvolutes these data using measurements of protein-drug affinity profiles. Through integrating a drug-kinase profiling dataset and several drug response datasets, our method, DepInfeR, correctly identified known protein kinase dependencies, including the EGFR dependence of HER2+ breast cancer cell lines, the FLT3 dependence of acute myeloid leukemia (AML) with FLT3-ITD mutations and the differential dependencies on the B-cell receptor pathway in the two major subtypes of chronic lymphocytic leukemia (CLL). Furthermore, our method uncovered new subgroup-specific dependencies, including a previously unreported dependence of high-risk CLL on Checkpoint kinase 1 (CHEK1). The method also produced a detailed map of the kinase dependencies in a heterogeneous set of 117 CLL samples. The ability to deconvolute polypharmacological phenotypes into underlying causal molecular dependencies should increase the utility of high-throughput drug response assays for functional precision oncology

Drug-perturbation-based stratification of blood cancer

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.Peer reviewe

High-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses.

A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA) with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV) 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV) of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA) based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R(2) = 0.7) was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively

Titration of different PSV preparations.

<p>Serial dilution of PSV preparations from HPV types 16, 18, 31, 45, 52, 58 and BPV-1 were assayed for Gaussia luciferase activity in the HT-PBNA. For HPV types 16 and 18 a second PSV preparation is indicated by an asterisk (*). Luminescence signals are expressed as relative light units (RLU). The titers for undiluted HPV 16, 18, and BPV-1 PSV were 3.0×10⁹, 3.9×10⁹, 1.1×10¹⁰ genomes per ml, respectively.</p

HT-PBNA inter- and intra-run variability of neutralization titers.

<p>The ED₅₀ values for HPV 16 and HPV 18 PSV of the serum standard were determined in 58 repeats on seven assay days (runs) over a period of 2 months. For six of the 7 assay days, triplicates of the standard serum dilutions were assayed 8 times each, for one assay date the standard serum was assayed 10 times.</p

Influence of PSV concentration on HT-PBNA analytical sensitivity and robustness.

<p>Neutralization titers of a serum from a Gardasil® immunized individual expressed as ED₅₀ values (open circles and open squares) with the variability (bars indicating the 95% confidence intervals) were determined at different PSV concentrations. The maximal luminescence intensities (RLU) obtained without serum are shown as crosses. An arrow indicates the dilution of the PSV preparation that was used in subsequent neutralization assays.</p

Effect of PSV-serum premix incubation time on neutralization titer.

<p>A serum from a Gardasil® vaccinated individual was pre-incubated for different times with HPV 16 or HPV 18 PSVs before the neutralization assay was initiated by the addition of reporter cells. ED₅₀ values with 95% confidence intervals are shown.</p

Analytical sensitivity and type-specificity of the HPV 16 and HPV 18 HT-PBNA.

<p>Titration of the WHO International Standards for antibodies to HPV 16 (left) and HPV 18 (right) in HT-PBNA using PSV of HPV types 16, 18, 31, 45, 52, 58 and BPV-1.</p

HT-PBNA protocol.

<p>Assay plate preparation (A) and neutralization assay (B) are separated. In a first step, serial dilutions of serum samples are performed on one dilution plate. Identical assay plates are generated by transferring the dilutions to multiple replica-assay plates which can be stored at −20°C. In the second step, the neutralization assay is carried out in a add-on format using the previously prepared assay plates. <b>A) Assay plate preparation</b>. Sera are transferred from a 96 well storage in SBS standard to a 384 well polypropylene V-bottom plate for serial dilution with a pipetting robot in cell culture medium. Finally the serially diluted sera are transferred with the same pipetting robot to each of 3*n white 384 well cell culture assay plates (n = number of PV-types). The plates are sealed immediately with a cover foil and stored at −20°C until their use in the PBNA. <b>B) Assay assembly and read out</b>. Assay plates are thawed and pseudovirions followed by reporter cells are added with a bulk dispenser. Three identical assay plates originating from the same serum dilution plate are used for each PV- type. After 2 days of incubation the luminescence from the Gaussia reporter is read directly in the assay plates. Inhibition (%) is calculated by normalization of the luminescence to the mean of the negative control wells without serum present on each plate. The median of the triplicate values is used for the calculation of the ED₅₀-value (effective dilution giving 50% inhibition) for each serum according to the four parameter logistic curve fit model y = A+(B−A)/(1+(C/x)^∧D).</p