Alun Parsons

Somatic STAT3 mutations in large granular lymphocytic leukemia.

BACKGROUND T-cell large granular lymphocytic leukemia is a rare lymphoproliferative disorder characterized by the expansion of clonal CD3+CD8+ cytotoxic T lymphocytes (CTLs) and often associated with autoimmune disorders and immune-mediated cytopenias. METHODS We used next-generation exome sequencing to identify somatic mutations in CTLs from an index patient with large granular lymphocytic leukemia. Targeted resequencing was performed in a well-characterized cohort of 76 patients with this disorder, characterized by clonal T-cell-receptor rearrangements and increased numbers of large granular lymphocytes. RESULTS Mutations in the signal transducer and activator of transcription 3 gene (STAT3) were found in 31 of 77 patients (40%) with large granular lymphocytic leukemia. Among these 31 patients, recurrent mutational hot spots included Y640F in 13 (17%), D661V in 7 (9%), D661Y in 7 (9%), and N647I in 3 (4%). All mutations were located in exon 21, encoding the Src homology 2 (SH2) domain, which mediates the dimerization and activation of STAT protein. The amino acid changes resulted in a more hydrophobic protein surface and were associated with phosphorylation of STAT3 and its localization in the nucleus. In vitro functional studies showed that the Y640F and D661V mutations increased the transcriptional activity of STAT3. In the affected patients, downstream target genes of the STAT3 pathway (IFNGR2, BCL2L1, and JAK2) were up-regulated. Patients with STAT3 mutations presented more often with neutropenia and rheumatoid arthritis than did patients without these mutations. CONCLUSIONS The SH2 dimerization and activation domain of STAT3 is frequently mutated in patients with large granular lymphocytic leukemia; these findings suggest that aberrant STAT3 signaling underlies the pathogenesis of this disease. (Funded by the Academy of Finland and others.).

Novel activating STAT5B mutations as putative drivers of T-cell acute lymphoblastic leukemia.

Novel activating STAT5B mutations as putative drivers of T-cell acute lymphoblastic leukemia

HOX gene expression predicts response to BCL-2 inhibition in acute myeloid leukemia

Inhibitors of B-cell lymphoma-2 (BCL-2) such as venetoclax (ABT-199) and navitoclax (ABT-263) are clinically explored in several cancer types, including acute myeloid leukemia (AML), to selectively induce apoptosis in cancer cells. To identify robust biomarkers for BCL-2 inhibitor sensitivity, we evaluated the ex vivo sensitivity of fresh leukemic cells from 73 diagnosed and relapsed/refractory AML patients, and then comprehensively assessed whether the responses correlated to specific mutations or gene expression signatures. Compared with samples from healthy donor controls (nonsensitive) and chronic lymphocytic leukemia (CLL) patients (highly sensitive), AML samples exhibited variable responses to BCL-2 inhibition. Strongest CLL-like responses were observed in 15% of the AML patient samples, whereas 32% were resistant, and the remaining exhibited intermediate responses to venetoclax. BCL-2 inhibitor sensitivity was associated with genetic aberrations in chromatin modifiers, WT1 and IDH1/IDH2. A striking selective overexpression of specific HOXA and HOXB gene transcripts were detected in highly BCL-2 inhibitor sensitive samples. Ex vivo responses to venetoclax showed significant inverse correlation to β2-microglobulin expression and to a lesser degree to BCL-XL and BAX expression. As new therapy options for AML are urgently needed, the specific HOX gene expression pattern can potentially be used as a biomarker to identify venetoclax-sensitive AML patients for clinical trials.

Activating somatic mutations outside the SH2-domain of STAT3 in LGL leukemia

Large granular lymphocyte (LGL) leukemia is a rare clonal disease characterized by a persistent increase in the number of CD8+ cytotoxic T cells or CD16/56+ natural killer (NK) cells.1 Patients are prone to recurrent infections and often suffer from severe cytopenias and autoimmune diseases that are thought to be mediated by cytotoxic LGL lymphocytes. LGL leukemia is believed to begin as an antigen-driven immune response followed by the constitutive activation of cytotoxic T lymphocytes or NK cells. Overall, studies have highlighted the dysregulation of different apoptotic pathways (for example, sphingolipid and FAS/FAS ligand) and the activation of survival signaling pathways (for example, PI3K/AKT and RAS).1

JAK1/2 and BCL2 inhibitors synergize to counteract bone marrow stromal cell-induced protection of AML

The bone marrow (BM) provides a protective microenvironment to support the survival of leukemic cells and influence their response to therapeutic agents. In acute myeloid leukemia (AML), the high rate of relapse may in part be a result of the inability of current treatment to effectively overcome the protective influence of the BM niche. To better understand the effect of the BM microenvironment on drug responses in AML, we conducted a comprehensive evaluation of 304 inhibitors, including approved and investigational agents, comparing ex vivo responses of primary AML cells in BM stroma-derived and standard culture conditions. In the stroma-based conditions, the AML patient cells exhibited significantly reduced sensitivity to 12% of the tested compounds, including topoisomerase II, B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL2), and many tyrosine kinase inhibitors (TKIs). The loss of TKI sensitivity was most pronounced in patient samples harboring FLT3 or PDGFRB alterations. In contrast, the stroma-derived conditions enhanced sensitivity to Janus kinase (JAK) inhibitors. Increased cell viability and resistance to specific drug classes in the BM stroma-derived conditions was a result of activation of alternative signaling pathways mediated by factors secreted by BM stromal cells and involved a switch from BCL2 to BCLXL-dependent cell survival. Moreover, the JAK1/2 inhibitor ruxolitinib restored sensitivity to the BCL2 inhibitor venetoclax in AML patient cells ex vivo in different model systems and in vivo in an AML xenograft mouse model. These findings highlight the potential of JAK inhibitors to counteract stroma-induced resistance to BCL2 inhibitors in AML.

Full-Genome Sequences of Influenza A(H1N1)pdm09 Viruses Isolated from Finnish Patients from 2009 to 2013

ABSTRACT Here we report full-length sequencing of the first large set of influenza A(H1N1)pdm09 virus genomes isolated in Finland between the years 2009 and 2013 and discuss the advantages and needs of influenza virus sequencing efforts.

Identification of precision treatment strategies for relapsed/ refractory multiple myeloma by functional drug sensitivity testing

Novel agents have increased survival of multiple myeloma (MM) patients, however high-risk and relapsed/refractory patients remain challenging to treat and their outcome is poor. To identify novel therapies and aid treatment selection for MM, we assessed the ex vivo sensitivity of 50 MM patient samples to 308 approved and investigational drugs. With the results we i) classified patients based on their ex vivo drug response profile; ii) identified and matched potential drug candidates to recurrent cytogenetic alterations; and iii) correlated ex vivo drug sensitivity to patient outcome. Based on their drug sensitivity profiles, MM patients were stratified into four distinct subgroups with varied survival outcomes. Patients with progressive disease and poor survival clustered in a drug response group exhibiting high sensitivity to signal transduction inhibitors. Del(17p) positive samples were resistant to most drugs tested with the exception of histone deacetylase and BCL2 inhibitors. Samples positive for t(4;14) were highly sensitive to immunomodulatory drugs, proteasome inhibitors and several targeted drugs. Three patients treated based on the ex vivo results showed good response to the selected treatments. Our results demonstrate that ex vivo drug testing may potentially be applied to optimize treatment selection and achieve therapeutic benefit for relapsed/refractory MM.Novel agents have increased survival of multiple myeloma (MM) patients, however high-risk and relapsed/refractory patients remain challenging to treat and their outcome is poor. To identify novel therapies and aid treatment selection for MM, we assessed the ex vivo sensitivity of 50 MM patient samples to 308 approved and investigational drugs. With the results we i) classified patients based on their ex vivo drug response profile; ii) identified and matched potential drug candidates to recurrent cytogenetic alterations; and iii) correlated ex vivo drug sensitivity to patient outcome. Based on their drug sensitivity profiles, MM patients were stratified into four distinct subgroups with varied survival outcomes. Patients with progressive disease and poor survival clustered in a drug response group exhibiting high sensitivity to signal transduction inhibitors. Del(17p) positive samples were resistant to most drugs tested with the exception of histone deacetylase and BCL2 inhibitors. Samples positive for t(4;14) were highly sensitive to immunomodulatory drugs, proteasome inhibitors and several targeted drugs. Three patients treated based on the ex vivo results showed good response to the selected treatments. Our results demonstrate that ex vivo drug testing may potentially be applied to optimize treatment selection and achieve therapeutic benefit for relapsed/refractory MM.

The impact of RNA sequence library construction protocols on transcriptomic profiling of leukemia

BackgroundRNA sequencing (RNA-seq) has become an indispensable tool to identify disease associated transcriptional profiles and determine the molecular underpinnings of diseases. However, the broad adaptation of the methodology into the clinic is still hampered by inconsistent results from different RNA-seq protocols and involves further evaluation of its analytical reliability using patient samples. Here, we applied two commonly used RNA-seq library preparation protocols to samples from acute leukemia patients to understand how poly-A-tailed mRNA selection (PA) and ribo-depletion (RD) based RNA-seq library preparation protocols affect gene fusion detection, variant calling, and gene expression profiling.ResultsOverall, the protocols produced similar results with consistent outcomes. Nevertheless, the PA protocol was more efficient in quantifying expression of leukemia marker genes and showed better performance in the expression-based classification of leukemia. Independent qRT-PCR experiments verified that the PA protocol better represented total RNA compared to the RD protocol. In contrast, the RD protocol detected a higher number of non-coding RNA features and had better alignment efficiency. The RD protocol also recovered more known fusion-gene events, although variability was seen in fusion gene predictions.ConclusionThe overall findings provide a framework for the use of RNA-seq in a precision medicine setting with limited number of samples and suggest that selection of the library preparation protocol should be based on the objectives of the analysis.

Drug sensitivity profiling identifies potential therapies for lymphoproliferative disorders with overactive JAK/STAT3 signaling

Constitutive JAK/STAT3 signaling contributes to disease progression in many lymphoproliferative disorders. Recent genetic analyses have revealed gain-of-function STAT3 mutations in lymphoid cancers leading to hyperactivation of STAT3, which may represent a potential therapeutic target. Using a functional reporter assay, we screened 306 compounds with selective activity against various target molecules to identify drugs capable of inhibiting the cellular activity of STAT3. Top hits were further validated with additional models including STAT3-mutated natural killer (NK)-cell leukemia/lymphoma cell lines and primary large granular lymphocytic (LGL) leukemia cells to assess their ability to inhibit STAT3 phosphorylation and STAT3 dependent cell viability. We identified JAK, mTOR, Hsp90 and CDK inhibitors as potent inhibitors of both WT and mutant STAT3 activity. The Hsp90 inhibitor luminespib was highly effective at reducing the viability of mutant STAT3 NK cell lines and LGL leukemia patient samples. Luminespib decreased the phosphorylation of mutant STAT3 at Y705, whereas JAK1/JAK2 inhibitor ruxolitinib had reduced efficacy on mutant STAT3 phosphorylation. Additionally, combinations involving Hsp90, JAK and mTOR inhibitors were more effective at reducing cell viability than single agents. Our findings show alternative approaches to inhibit STAT3 activity and suggest Hsp90 as a therapeutic target in lymphoproliferative disorders with constitutively active STAT3.

Abstract 3175: Genomic and transcriptomic data integration in chronic myelomonocytic leukemia reveals a novel fusion gene involving onco-miR-125b-2

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Chronic myelomonocytic leukemia (CMML) is a rare malignancy characterized by increased peripheral monocytosis and dysplasia in a single- or multilineage fashion. Gene mutations so far reported in CMML include TET2, CBL, NRAS, KRAS, RUNX1 and EZH2 but their pathogenic role and driver status in the disease remains unclear. Altered expression of the microRNA miR-125b has been implicated in the pathogenesis of many types of cancers, including myeloid leukemias and Down syndrome-associated acute myeloid leukemia (DS-AML). In addition, this miRNA has been shown to play an important role in hematopoiesis and the regulation of immune cell response. Here, integration of data from next-generation transcriptome sequencing, exome sequencing and array-CGH in a CMML patient (trisomy 21 by cytogenetics) led to the identification of a novel gene fusion event involving the nuclear receptor interacting protein NRIP1 gene and the open reading frame C21orf34 (both at 21q21 approximately 1 MB apart). The fusion was validated by capillary sequencing and found to involve two copy number transition breaks, inversion of the intervening region and the upregulation of the 3′ end of C21orf34. This intronic region harbors a cluster of three miRNAs: miR-let7c, miR-99a, and miR-125b-2. Based on genomic breakpoint analysis, the gene fusion took place just upstream of miR-125b-2. Consistent with this, only miR-125b-2 was highly expressed in the sample, and was processed to a mature miRNA. By RT-PCR, increased expression of miR-125b-2 was also observed in four other CMML patients and five CML patients when compared to healthy bone marrow controls. In contrast, five AML cases studied showed expression levels similar to or lower than that of controls. Interestingly, one AML patient with trisomy 21 had very high levels of miR-125b-2. We found the NRIP1-C21orf34 fusion only in our index patient and therefore other mechanisms of miRNA deregulation at 21q21 in CMML/CML and AML+21 will also exist. In conclusion, we describe for the first time a fusion gene involving miR-125b-2 in CMML, a previously recognized and well-studied onco-miR, which is known to impact on self-renewal of hematopoietic cell precursors. We also detected overexpression of miR-125b-2 in all CMML samples studied suggesting a key pathogenetic driver gene role for this micro-RNA. The assessment of miR-125b-2 levels could potentially be applied to the diagnosis and follow-up of patients with CMML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3175. doi:1538-7445.AM2012-3175