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.
Introduction
T-PLL is a mature post-thymic T-cell neoplasm with an aggressive clinical course (5-year overall survival 21%). Almost 75% of T-PLL cases harbor chromosome 14 translocations resulting in ...aberrant activation of the proto-oncogene TCL1A. Furthermore, in the majority of T-PLL cases the ATM gene is mutated or deleted, and recently it was reported that mutations in genes involved in the JAK-STAT pathway were found in 76% of T-PLL cases. Due to the rareness and aggressive nature of the disease, clinical trials are difficult to execute. By using a high-throughput ex vivo drug sensitivity and resistance testing (DSRT) platform covering 306 approved and investigational oncology drugs we systematically investigated the heterogeneity of drug responses in PLL-patients. As the impact of mutations on drug sensitivity is not well understood we aimed to identify relevant associations between the drug responses and genetic lesions in T-PLL patients.
Methods
Primary cells (MNCs) from seven T-PLL patients were obtained for drug screening. Samples were seeded in 384-well plates and 306 active substances were tested using a 10,000-fold concentration range resulting in a dose-response curve for each compound. Cell viability was measured after 72 h incubation and differential drug sensitivity scores (sDSS), representing leukemia-specific responses, were calculated by comparing patient samples to healthy donors. Hierarchical clustering of the drug responses was performed with Cluster 3.0 and Java Tree View. To assess the performance of the drug screening platform we also exchanged six samples with the German Cancer Research Center in Heidelberg for a comparison of results between two independent drug screening systems.
To understand heterogeneous pathway dependencies, drug sensitivities were correlated with somatic genetic variants and recurrent chromosomal aberrations. Genetic characterization was performed by exome sequencing of tumor and matched healthy cells to profile known recurrent genetic variants (ATM, STAT5b, IL2RG, JAK1, JAK3) as well as CNVs (TCL1A translocations, ATM deletions, recurrent chromosomal aberrations).
Results
Four out of seven patient samples showed high sensitivity to small molecule BCL2 inhibitors navitoclax (IC50: 10-68nM) and ABT-199 (IC50: 14-45nM) and to HDAC inhibitors panobinostat and belinostat (IC50: 2-65nM). Intriguingly, the CDK inhibitor SNS-032 was effective in 6/7 patient samples (IC50: 7-95nM). SNS-032 inhibits Cdk2, Cdk7 and Cdk9, which control transcription of anti-apoptotic proteins including MCL1 and XIAP. As the AKT1/MTOR pathway is activated in many T-PLL patients due to expression of the TCL1A oncoprotein, it was interesting to observe that patient samples did not show any response to AKT inhibitors (MK-2206 and GDC-0068 IC50 values >1000 nM) nor to MTOR inhibitors (rapalogs temsirolimus and everolimus). Similarly, T-PLL cells were insensitive to JAK-inhibitors.
Clustering of drug responses from T-PLL patients with primary AML and ALL patient samples revealed the drug response profiles to be specific for T-PLL patients (Figure). 6/7 patients clustered together while the only patient (PLL4) in our cohort with confirmed mutations in the JAK-STAT pathway genes STAT5b (P702S) and IL2RG (K315E) exhibited a non-sensitive response pattern when compared to other samples (Figure). Interestingly, exome sequencing did not reveal any JAK mutations in our PLL-cohort (n=5) nor additional STAT5b or IL2RG mutations in other patients except in this unresponsive patient.
In the comparison between the platforms the correlation of the censored IC50 values from the 60 overlapping drugs was r=0.75. Similar fits of dose-response curves were seen for most drugs, although there were notable exceptions, which may be due to divergent culture conditions and day of read-out.
Conclusions
Ex vivo drug testing of primary patient cells has the potential to provide novel personalized drug candidates (such as BCL2, HDAC and CDK inhibitors) for T-PLL. The drug response pattern was T-PLL specific warranting further clinical testing. Drug screening, mutation analysis and RNA sequencing of additional patients is currently ongoing (n=20) to validate whether drug responses can be predicted based on the mutation profile or aberrant gene expression.
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Kallioniemi:Medisapiens: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.
This paper develops a regional production function model for Swiss cantons that incorporates human capital together with spatial effects. Within a spatial panel framework we find that controlling for ...time effects the spatial spillover effect becomes insignificant. Our results are sensitive with respect to the human capital proxy. We find that the share of academics in the workforce is the main component of human capital driving productivity growth in Swiss cantons. This is in line with findings of previous studies suggesting that mostly highly skilled workers matter for productivity growth in technologically advanced economies.
Inhibition of the aspartyl protease renin is considered as an efficient approach for treating hypertension. Lately, we described the discovery of a novel class of direct renin inhibitors which ...comprised a pyrrolidine scaffold (e.g., 2). Based on the X-ray structure of the lead compound 2 bound to renin we predicted that optimization of binding interactions to the prime site could offer an opportunity to further expand the scope of this chemotype. Pyrrolidine-based inhibitors were synthesized in which the prime site moieties are linked to the pyrrolidine core through an oxygen atom, resulting in an ether or a carbamate linker subseries. Especially the carbamate derivatives showed a pronounced increase in in vitro potency compared to 2. Here we report the structure-activity relationship of both subclasses and demonstrate blood pressure lowering effects for an advanced prototype in a hypertensive double-transgenic rat model after oral dosing.
Various approaches can now be taken for amplification of RNA transcripts using the polymerase chain reaction (PCR). Here, we compare three such methods: (i) uncoupled reverse transcription (RT)-PCR ...(using separate reactions for cDNA synthesis and PCR), (ii) continuous RT-PCR (in which RT and DNA amplification occur in an uninterrupted reaction) using either a single enzyme for both RT and DNA amplification or (iii) using two enzymes, one for each task. We have found that the continuous two-enzyme RT-PCR method is the most sensitive, followed by the uncoupled RT-PCR and then the continuous single-enzyme method. The continuous methods require less sample handling than the uncoupled method, and hence are less labor-intensive and less prone to contamination. The continuous single-enzyme method is the most expensive to perform in terms of reagents due to the quantity of DNA polymerase required; however, it does have advantages over the two enzyme methods in that the use of a thermostable enzyme for RT can alleviate certain problems by allowing RT to occur at higher temperatures than those tolerable by viral reverse transcriptases.
T-cell prolymphocytic leukemia (T-PLL) is a rare disease with an aggressive clinical course and a median overall survival of less than three years. Although almost 75% of T-PLL patients are reported ...to harbor translocations causing the activation of the proto-oncogene TCL1A, T-PLL is genetically heterogenous: most T-PLL patients also have mutations or deletions in the ATM gene and the genes involved in the JAK-STAT pathway are mutated in 76% of cases. There is an urgent need for more rational based therapies, but clinical trials are difficult to perform due to the rareness of the disease. Here, we systematically explored the diversity of drug responses in T-PLL patient samples ex vivo using a drug sensitivity and resistance testing (DSRT) system including 306 oncology drugs (approved or investigational). We also aimed to determine any associations between the genetic aberrations and drug sensitivities in T-PLL patients.
Primary mononuclear cells were gathered from 30 T-PLL patients for drug testing. Cells were plated in 384-well plates and subjected to the 306 substances using a 10,000-fold concentration range. After 72 hours, cell viabilities were measured, the results were depicted as dose-response curves for each compound, and differential drug sensitivity scores (sDSS), representing leukemia-specific responses, were computed by comparing patient samples to healthy donors. Drug response profiles across patients were clustered and visualized by hierarchical clustering. The subgroups resulting from the clustering were statistically compared using a two-sample t-test to understand which drug classes were driving the grouping.
To delineate heterogeneous pathway dependencies, drug sensitivities were correlated with somatic genetic variants and recurrent chromosomal aberrations. Genetic characterization was performed by targeted amplicon sequencing of tumor cells to profile known recurrent genetic variants (STAT5b, IL2RG, JAK1, JAK3, ATM). Information on chromosomal aberrations (TCL1A translocations, ATM deletions) was derived from parallel clinicopathologic databases.
Amplicon sequencing revealed that 70% of T-PLL patients (21/30) harbored a mutation in genes involved in the JAK-STAT pathway (JAK1, JAK3, STAT5b or IL2RG). The most prevalent mutation led to an M511I amino acid exchange in the JAK3 protein (26% of patients). Interestingly, the STAT5b mutations (5/30) did not coexist with any of the JAK mutations in our cohort.
Based on DSRT analysis, all T-PLL samples were sensitive to the CDK-inhibitor SNS-032 and the anti-cancer antibiotic actinomycin D. Next, we clustered patients using sDSS values for all 306 different substances, and this showed that patient samples could be divided into 3 main groups, based on their drug responses (Figure). According to two-sample t-test, the grouping was driven by the selective sensitivities of Groups II and III to HDAC inhibitors (belinostat, panobinostat, quisinostat, CUDC-101 and vorinostat) and the selective sensitivity of Group III to PI3K/AKT/mTOR inhibitors (AZD-8055, MK-2206, apitolisib, dactolisib, PF-04691502, ZSTK474, and omipalisib), HSP90 inhibitors (BIIB021, luminespib, alvespimycin, and tanespimycin) as well as JAK inhibitors (ruxolitinib, momelotinib, tofacitinib, gandotinib). Group I samples were on the other hand relatively resistant to these classes of drugs. Surprisingly, despite the prevalence of the signature event of activation of TCL1 (the established AKT coactivator) in nearly all cases, only a subset of cases (group III) responded to PI3K/AKT/mTOR inhibitors. Strikingly, the grouping of selective responses to HDAC, JAK, PI3K/mTOR/Akt and HSP90 inhibitors did not link to the presence of JAK/STAT mutations, TCL1A translocations, or ATM deletion status.
Ex vivo drug screening of primary T-PLL samples revealed heterogenous selective drug responses in specific drug classes (such as HDAC-, JAK-, HSP90- and PI3K/Akt/mTOR-inhibitors). Surprisingly, the drug response patterns did not correlate with known recurrent genetic aberrations suggesting that sequencing for recurrent genetic biomarkers cannot easily be turned into effective therapeutic strategies in T-PLL, and that further elucidation of the biological pathways driving T-PLL is needed.
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Koschmieder:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences, Research Funding; Novartis Foundation: Research Funding; Baxalta/CTI: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Other: Travel reimbursement for scientific conferences ; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences. Wennerberg:Pfizer: Honoraria, Research Funding. Ding:Merek: Research Funding. Mustjoki:Pfizer: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.
High-precision comparisons of the proton-to-antiproton charge-to-mass ratios provide sensitive tests of the fundamental charge, parity, time (CPT) invariance. In 2014, we performed such a measurement ...with a fractional precision of 69parts in a trillion (p.p.t.). In this article, we describe technical developments which were implemented to improve the precision of our previous measurement by at least a factor of 3.
We present an experimental study on the first stages of the thin film growth of the organic molecule F16CuPc (hexadecafluoro-copper-phthalocyanines) on SiO2. By means of in situ X-ray reflectivity, ...in situ grazing incidence X-ray diffraction (GIXD), and ex situ atomic force microscopy (AFM), we provide a detailed picture of the film growth mode and its structural evolution at the nanometer scale. We discovered the formation of a low-density layer of molecular aggregates with heights between 5 and 10 Å at the interface with the SiO2 and show that, on top of this interfacial layer, the nucleation and two-dimensional growth of elongated islands of upright standing molecules take place. Structural changes are observed, pointing to significant relaxations of the lattice parameters within the first layers of standing molecules.
Chronic lymphocytic leukemia (CLL) shows a heterogeneous clinical course, which can be explained in part by prognostic factors. Most patients do not need treatment at the time of first diagnosis. The ...identification of prognostic factors is of major interest if strategies can be devised to treat patients according to their individual risk profile or biological subgroup. Currently, in spite of a wealth of prognostic factors, individualized treatment approaches in different genetic or risk groups are the exemption in CLL. This review summarizes the most important prognostic and predictive factors in CLL, with particular emphasis on factors affecting treatment decisions in clinical trials and routine practice.
Incidental brain magnetic resonance imaging (MRI) findings are the result of an increasing usage of MRI in the diagnostic work-up of patients. An adequate assessment of patients in which brain ...lesions typical for multiple sclerosis (MS) are determined but who have been asymptomatic so far is problematic, especially when Barkhof-Tintoré criteria for spatial dissemination are fulfilled and no other differential diagnosis can be confirmed. This entity, the so-called radiologically isolated syndrome, constitutes a major diagnostic and therapeutic challenge. Two recent studies revealed that a subgroup of patients with radiologically isolated syndrome are at high risk for near-term development of MR-based progression and occurrence of the first clinical event. Hence, the radiologically isolated syndrome has to be classified as a possible preliminary phase of the clinical manifestation of MS in a subgroup of patients and entails in-depth therapeutic considerations. This article covers the current literature for this syndrome and, in the absence of official guidelines, provides a pragmatic diagnostic and therapeutic approach for patient management.
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