Arjan J. van Adrichem

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.).

Discovery of somatic STAT5b mutations in large granular lymphocytic leukemia

Large granular lymphocytic (LGL) leukemia is characterized by clonal expansion of cytotoxic T cells or natural killer cells. Recently, somatic mutations in the signal transducer and activator of transcription 3 (STAT3) gene were discovered in 28% to 40% of LGL leukemia patients. By exome and transcriptome sequencing of 2 STAT3 mutation-negative LGL leukemia patients, we identified a recurrent, somatic missense mutation (Y665F) in the Src-like homology 2 domain of the STAT5b gene. Targeted amplicon sequencing of 211 LGL leukemia patients revealed 2 additional patients with STAT5b mutations (N642H), resulting in a total frequency of 2% (4 of 211) of STAT5b mutations across all patients. The Y665F and N642H mutant constructs increased the transcriptional activity of STAT5 and tyrosine (Y694) phosphorylation, which was also observed in patient samples. The clinical course of the disease in patients with the N642H mutation was aggressive and fatal, clearly different from typical LGL leukemia with a relatively favorable outcome. This is the first time somatic STAT5 mutations are discovered in human cancer and further emphasizes the role of STAT family genes in the pathogenesis of LGL leukemia.

MiR-17/20/93/106 promote hematopoietic cell expansion by targeting sequestosome 1–regulated pathways in mice

MicroRNAs (miRNAs) are pivotal for regulation of hematopoiesis but their critical targets remain largely unknown. Here, we show that ectopic expression of miR-17, -20,-93 and -106, all AAAGUGC seed-containing miRNAs, increases proliferation, colony outgrowth and replating capacity of myeloid progenitors and results in enhanced P-ERK levels. We found that these miRNAs are endogenously and abundantly expressed in myeloid progenitors and down-regulated in mature neutrophils. Quantitative proteomics identified sequestosome 1 (SQSTM1), an ubiquitin-binding protein and regulator of autophagy-mediated protein degradation, as a major target for these miRNAs in myeloid progenitors. In addition, we found increased expression of Sqstm1 transcripts during CSF3-induced neutrophil differentiation of 32D-CSF3R cells and an inverse correlation of SQSTM1 protein levels and miR-106 expression in AML samples. ShRNA-mediated silencing of Sqstm1 phenocopied the effects of ectopic miR-17/20/93/106 expression in hematopoietic progenitors in vitro and in mice. Further, SQSTM1 binds to the ligand-activated colony-stimulating factor 3 receptor (CSF3R) mainly in the late endosomal compartment, but not in LC3 positive autophagosomes. SQSTM1 regulates CSF3R stability and ligand-induced mitogen-activated protein kinase signaling. We demonstrate that AAAGUGC seed-containing miRNAs promote cell expansion, replating capacity and signaling in hematopoietic cells by interference with SQSTM1-regulated pathways.

Drug Target Commons: A Community Effort to Build a Consensus Knowledge Base for Drug-Target Interactions

Summary Knowledge of the full target space of bioactive substances, approved and investigational drugs as well as chemical probes, provides important insights into therapeutic potential and possible adverse effects. The existing compound-target bioactivity data resources are often incomparable due to non-standardized and heterogeneous assay types and variability in endpoint measurements. To extract higher value from the existing and future compound target-profiling data, we implemented an open-data web platform, named Drug Target Commons (DTC), which features tools for crowd-sourced compound-target bioactivity data annotation, standardization, curation, and intra-resource integration. We demonstrate the unique value of DTC with several examples related to both drug discovery and drug repurposing applications and invite researchers to join this community effort to increase the reuse and extension of compound bioactivity data.

Discovery of MINC1, a GTPase-Activating Protein Small Molecule Inhibitor, Targeting MgcRacGAP

The Rho family of Ras superfamily small GTPases regulates a broad range of biological processes such as migration, differentiation, cell growth and cell survival. Therefore, the availability of small molecule modulators as tool compounds could greatly enhance research on these proteins and their biological function. To this end, we designed a biochemical, high throughput screening assay with complementary follow-up assays to identify small molecule compounds inhibiting MgcRacGAP, a Rho family GTPase activating protein involved in cytokinesis and transcriptionally upregulated in many cancers. We first performed an in-house screen of 20,480 compounds, and later we tested the assay against 342,046 compounds from the NIH Molecular Libraries Small Molecule Repository. Primary screening hit rates were about 1% with the majority of those affecting the primary readout, an enzyme-coupled GDP detection assay. After orthogonal and counter screens, we identified two hits with high selectivity towards MgcRacGAP, compared with other RhoGAPs, and potencies in the low micromolar range. The most promising hit, termed MINC1, was then examined with cell-based testing where it was observed to induce an increased rate of cytokinetic failure and multinucleation in addition to other cell division defects, suggesting that it may act as an MgcRacGAP inhibitor also in cells.

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.

MgcRacGAP inhibition stimulates JAK-dependent STAT3 activity.

Male germ cell Rac GTPase‐activating protein (MgcRacGAP) is a core regulator of cytokinesis. Furthermore, it appears to be involved in human oncogenesis through cytokinesis‐independent mechanisms and has been reported to be essential for nuclear translocation of signal transducer and activator of transcription (STAT) proteins, including the oncoprotein STAT3. Here we utilized MgcRacGAP inhibitor compound 1 (MINC1), a small molecule inhibitor of MgcRacGAP, to further investigate how MgcRacGAP regulates STAT3. Surprisingly, both MINC1 treatment and small interference RNA (siRNA)‐mediated gene silencing of MgcRacGAP resulted in increased STAT3 phosphorylation and STAT3‐driven transcriptional activity in our experimental systems. Finally, we demonstrated that MINC1‐induced STAT3 activation likely is due to increased STAT3 phosphorylation caused by a Rac1‐PAR3‐IL6‐IL6R‐JAK2 mediated autocrine/paracrine mechanism.

Abstract 2991: Inhibition of Rac1 GTPase activity by SH7139, a new drug candidate for non-Hodgkin's lymphoma targeting HLA-DR10

SH7139, the first in a series of selective high affinity ligand (SHAL) therapeutics designed to treat non-Hodgkin9s lymphoma, has been shown to be selectively cytotoxic to lymphoma cells over-expressing HLA-DR10. Recent efforts to elucidate the mechanisms of action of SH7139 show that the small molecule drug functions similar to both an antibody drug conjugate and a pro-drug. SH7139 is comprised of three small molecule recognition elements that, when linked together, collectively target the drug to HLA-DR10. Following its binding to HLA-DR10, SH7139 is shuttled into the interior of the lymphoma cell where the subsequent metabolism of these recognition elements releases a series of metabolites that inhibit multiple activities required for tumor cell growth and replication. Studies performed using the Burkitt9s lymphoma cell line Raji have shown that SH7139 is metabolized by Raji cells, and the metabolic cleavage of two of the recognition elements (Ct and Dv) produce cytotoxic compounds that contribute to tumor cell killing. While the third recognition element, Cb, is not cleaved off the SHAL scaffold or hydrolyzed to release a cytotoxic metabolite, its structural similarity to known inhibitors of the GTPase activating protein (GAP) MgcRacGAP suggested that it might be active in this pathway. MgcRacGAP functions as a switch that stimulates by many orders of magnitude the activity of the Rac1 GTPase, which is required for cleavage furrow formation, ingression, and the completion of cytokinesis. Experiments conducted with the MgcRacGAP-Rac1 complex have shown that the intact SH7139 molecule (IC50 = 10.6±1.6μM) as well as SH7139 fragments containing the Cb recognition element is effective in inhibiting the GTPase activity of the MgcRacGAP:Rac1 complex. These results confirm that one mechanism action of SH7139 is the inhibition of the Rac1-dependent effector pathways that control the rounding of cells undergoing mitosis, confine Rho activation to the equator of the cell for proper cleavage furrow formation and other processes involved in the completion of cytokinesis. This research was supported by the National Cancer Institute Phase II SBIR award R44CA159843 to SHAL Technologies Inc. Part of this work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. Citation Format: Rodney Balhorn, Arjan J. van Adrichem, Saphon Hok, Monique C. Balhorn. Inhibition of Rac1 GTPase activity by SH7139, a new drug candidate for non-Hodgkin9s lymphoma targeting HLA-DR10. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2991.

Abstract 606: Novel somatic mutations in the DNA-binding and coiled-coil domain of the STAT3 gene in LGL-leukemia

Introduction T-cell large granular lymphocyte (T-LGL) leukemia is a rare, clonal disease characterized by the expansion of CD8+ cytotoxic T-cells. We recently discovered that 40% of T-LGL leukemia patients have somatic mutations in the SH2-domain of the STAT3 gene (Koskela et al. NEJM 2012). As aberrant STAT3 activation can be observed in all patients with LGL-leukemia, we now aimed to discover whether patients without mutations in the STAT3 hotspot area harbor mutations in the other parts of the STAT3 gene. Methods Targeted STAT3 sequencing covering all 23 coding exons was done with in-house developed deep amplicon sequencing panel using the Illumina Miseq platform. The data was analyzed with a bioinformatics pipeline, which is based on calling of variants with specific counts/frequencies and filtering out false positives using the estimated error rate and quality data of amplicon reads. All samples with a frequency ratio ≥0.9 were considered to be true mutations after filtering of SNPs and low coverage variants. 111 LGL-leukemia patients with no known STAT3-mutations in the SH2 domain were analyzed. To explore the functional effects of mutations, expression constructs were generated with the identified variants and wild-type STAT3. The variants were expressed in HEK-293 cells carrying a STAT3-responsive SIE-reporter driven luciferase expression sequence to establish increased basal and IL6-stimulated STAT3 activity. Results With targeted amplicon sequencing, 3 patients were discovered to have STAT3 missense mutations in the DNA-binding domain. Two patients presented with the same H410R mutation with a variant allele frequency (VAF) of 49% and 8.8% respectively while another patient had a S381Y mutation (VAF 7%). The mutation H410R occurs in the DNA-binding domain in a highly conserved position, and results in conversion of histidine to arginine, which would predict for a slight increase in hydrophilicity. In addition to STAT3 DNA-binding domain mutations, one T-LGL patient had a novel F174S mutation in the coiled-coil domain of STAT3 (VAF 43%). The coiled-coil domain of STAT3 has previously been shown to be essential in SH2-domain mediated receptor binding and subsequent activation. Luciferase measurements of SIE-reporter HEK-293 cells transfected with constructs expressing either wild-type, variant F174S, H410R or Y640F STAT3 (the most common activating mutation in LGL leukemia) revealed the F174S and H410R variants to be as activating as the Y640F mutation in both unstimulated and IL-6 stimulated conditions. Conclusions T-LGL leukemia patients without STAT3 SH2-domain mutations harbor novel activating mutations in the DNA-binding and coiled-coil domain of STAT3. The frequency of mutations was 3.6% (4 of 111 patients). These findings further highlight the importance of screening the whole STAT3 gene in the diagnostic workup of LGL-leukemia and the central role of STAT3 in the pathogenesis of the disease. Citation Format: Emma I. Andersson, Hanna Rajala, Heikki Kuusanmaki, Arjan van Adrichem, Samuli Eldfors, Sonja Lagstrom, Thomas Olson, Michael Clemente, Pekka Ellonen, Caroline Heckman, Thomas P. Loughran, Jaroslaw P. Maciejewski, Satu Mustjoki. Novel somatic mutations in the DNA-binding and coiled-coil domain of the STAT3 gene in LGL-leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 606. doi:10.1158/1538-7445.AM2015-606