Identification of novel regulators of STAT3 activity

STAT3 mediates signalling downstream of cytokine and growth factor receptors where it acts as a transcription factor for its target genes, including oncogenes and cell survival regulating genes. STAT3 has been found to be persistently activated in many types of cancers, primarily through its tyrosine phosphorylation (Y705). Here, we show that constitutive STAT3 activation protects cells from cytotoxic drug responses of several drug classes. To find novel and potentially targetable STAT3 regulators we performed a kinase and phosphatase siRNA screen with cells expressing either a hyperactive STAT3 mutant or IL6-induced wild type STAT3. The screen identified cell division cycle 7-related protein kinase (CDC7), casein kinase 2, alpha 1 (CSNK2), discoidin domain-containing receptor 2 (DDR2), cyclin-dependent kinase 8 (CDK8), phosphatidylinositol 4-kinase 2-alpha (PI4KII), C-terminal Src kinase (CSK) and receptor-type tyrosine-protein phosphatase H (PTPRH) as potential STAT3 regulators. Using small molecule inhibitors targeting these proteins, we confirmed dose and time dependent inhibition of STAT3-mediated transcription, suggesting that inhibition of these kinases may provide strategies for dampening STAT3 activity in cancers.Peer reviewe

Phenotype-based drug screening reveals association between venetoclax response and differentiation stage in acute myeloid leukemia

Ex vivo drug testing is a promising approach to identify novel treatment strategies for acute myeloid leukemia (AML). However, accurate blast- specific drug responses cannot be measured with homogeneous "add-mix-measure" cell viability assays. In this study, we implemented a flow cytometry-based approach to simultaneously evaluate the ex vivo sensitivity of different cell populations in 34 primary AML samples to seven drugs and 27 rational drug combinations. Our data demonstrate that different cell populations present in AML samples have distinct sensitivity to targeted therapies. Particularly, blast cells of FAB M0/1 AML showed high sensitivity to venetoclax. In contrast, differentiated monocytic cells abundantly present in M4/5 subtypes showed resistance to Bcl-2 inhibition, whereas immature blasts in the same samples were sensitive, highlighting the importance of blast-specific readouts. Accordingly, in the total mononuclear cell fraction the highest BCL2/MCL1 gene expression ratio was observed in M0/1 and the lowest in M4/5 AML. Of the seven tested drugs, venetoclax had the highest blast-specific toxicity, and combining venetoclax with either MEK inhibitor trametinib or JAK inhibitor ruxolitinib effectively targeted all venetoclax-resistant blasts. In conclusion, we show that ex vivo efficacy of targeted agents and particularly Bcl-2 inhibitor venetoclax is influenced by the cell type, and accurate blast-specific drug responses can be assessed with a flow cytometry-based approach.Peer reviewe

Somatic MED12 Nonsense Mutation Escapes mRNA Decay and Reveals a Motif Required for Nuclear Entry

MED12 is a key component of the transcription-regulating Mediator complex. Specific missense and in-frame insertion/deletion mutations in exons 1 and 2 have been identified in uterine leiomyomas, breast tumors, and chronic lymphocytic leukemia. Here, we characterize the first MED12 5 end nonsense mutation (c.97G > T, p.E33X) identified in acute lymphoblastic leukemia and show that it escapes nonsense-mediated mRNA decay (NMD) by using an alternative translation initiation site. The resulting N-terminally truncated protein is unable to enter the nucleus due to the lack of identified nuclear localization signal (NLS). The absence of NLS prevents the mutant MED12 protein to be recognized by importin- and subsequent loading into the nuclear pore complex. Due to this mislocalization, all interactions between the MED12 mutant and other Mediator components are lost. Our findings provide new mechanistic insights into the MED12 functions and indicate that somatic nonsense mutations in early exons may avoid NMD. (C) 2017 Wiley Periodicals, Inc.Peer reviewe

Implementing a Functional Precision Medicine Tumor Board for Acute Myeloid Leukemia

We generated ex vivo drug-response and multiomics profi ling data for a prospective series of 252 samples from 186 patients with acute myeloid leukemia (AML). A functional precision medicine tumor board (FPMTB) integrated clinical, molecular, and functional data for application in clinical treatment decisions. Actionable drugs were found for 97% of patients with AML, and the recommendations were clinically implemented in 37 relapsed or refractory patients. We report a 59% objective response rate for the individually tailored therapies, including 13 complete responses, as well as bridging five patients with AML to allogeneic hematopoietic stem cell transplantation. Data integration across all cases enabled the identifi cation of drug response biomarkers, such as the association of IL15 overexpression with resistance to FLT3 inhibitors. Integration of molecular profi ling and large-scale drug response data across many patients will enable continuous improvement of the FPMTB recommendations, providing a paradigm for individualized implementation of functional precision cancer medicine. SIGNIFICANCE: Oncogenomics data can guide clinical treatment decisions, but often such data are neither actionable nor predictive. Functional ex vivo drug testing contributes signifi cant additional, clinically actionable therapeutic insights for individual patients with AML. Such data can be generated in four days, enabling rapid translation through FPMTB.Peer reviewe

Implementing a Functional Precision Medicine Tumor Board for Acute Myeloid Leukemia

We generated ex vivo drug-response and multiomics profi ling data for a prospective series of 252 samples from 186 patients with acute myeloid leukemia (AML). A functional precision medicine tumor board (FPMTB) integrated clinical, molecular, and functional data for application in clinical treatment decisions. Actionable drugs were found for 97% of patients with AML, and the recommendations were clinically implemented in 37 relapsed or refractory patients. We report a 59% objective response rate for the individually tailored therapies, including 13 complete responses, as well as bridging five patients with AML to allogeneic hematopoietic stem cell transplantation. Data integration across all cases enabled the identifi cation of drug response biomarkers, such as the association of IL15 overexpression with resistance to FLT3 inhibitors. Integration of molecular profi ling and large-scale drug response data across many patients will enable continuous improvement of the FPMTB recommendations, providing a paradigm for individualized implementation of functional precision cancer medicine. SIGNIFICANCE: Oncogenomics data can guide clinical treatment decisions, but often such data are neither actionable nor predictive. Functional ex vivo drug testing contributes signifi cant additional, clinically actionable therapeutic insights for individual patients with AML. Such data can be generated in four days, enabling rapid translation through FPMTB.Peer reviewe

Aggressive natural killer-cell leukemia mutational landscape and drug profiling highlight JAK-STAT signaling as therapeutic target

Aggressive natural killer-cell (NK-cell) leukemia (ANKL) is an extremely aggressive malig- nancy with dismal prognosis and lack of targeted therapies. Here, we elucidate the molecular pathogenesis of ANKL using a combination of genomic and drug sensitivity profiling. We study 14 ANKL patients using whole-exome sequencing (WES) and identify mutations in STAT3 (21%) and RAS-MAPK pathway genes (21%) as well as in DDX3X (29%) and epi- genetic modifiers (50%). Additional alterations include JAK-STAT copy gains and tyrosine phosphatase mutations, which we show recurrent also in extranodal NK/T-cell lymphoma, nasal type (NKTCL) through integration of public genomic data. Drug sensitivity profiling further demonstrates the role of the JAK-STAT pathway in the pathogenesis of NK-cell malignancies, identifying NK cells to be highly sensitive to JAK and BCL2 inhibition compared to other hematopoietic cell lineages. Our results provide insight into ANKL genetics and a framework for application of targeted therapies in NK-cell malignancies.Aggressiivinen NK-soluleukemia (ANKL) on elimistön luonnolliseen puolustusjärjestelmään kuuluvien luonnollisten tappajasolujen eli natural killer (NK) –solujen verisyöpä eli leukemia. ANKL:aan sairastuneet potilaat säilyvät käytössä olevilla solunsalpaaja- ja kantasolusiirtohoidoilla elossa keskimäärin vain joitakin kuukausia. Erityisesti aasialaisväestössä esiintyvän ANKL:n lisäksi NK-soluisiin syöpiin kuuluu Suomessakin harvinaisina tavattavia NK/T-soluisia lymfoomia. ANKL:n taustalla olevia hankittuja geenimuutoksia eli mutaatioita ei ole aiemmin selvitetty laajamittaisesti. Tutkimuksessa selvitimme ANKL:n tautimekanismeja kartoittamalla 14 potilaan syöpäsolujen geenimuutokset perimän proteiineja koodaavien geenien osalta ja tutkimalla pahanlaatuisten NK-solujen herkkyyttä yli 400 lääkeaineelle. Löysimme ANKL-potilaiden soluista geenimuutoksia etenkin STAT3- ja DDX3X-geeneissä, joita kumpiakin oli yli viidenneksellä potilaista. STAT3-mutaatioita on aiemmin todettu suurten granulaaristen lymfosyyttien (LGL) leukemiassa, sekä useissa muissakin T- ja NK-soluista lähtöisin olevissa syövissä. STAT3-mutaatiot ANKL:ssa viittaavat osin yhteisiin tautimekanismeihin näiden sukulaistautien kanssa. Kun yhdistimme ANKL-potilaiden geenitietoa aiemmin julkaistujen NK//T-solulymfoomapotilaista tuotettujen aineistojen kanssa, havaitsimme NK-soluisille syöville yhteisiä JAK-STAT-signalointigeenien monistumia. Etsimme myös potentiaalisia lääkeaineita NK-soluisten syöpien hoitoon testaamalla pahanlaatuisten NK-solujen herkkyyttä yli 400 lääkeaineelle. Havaitsimme NK-solujen olevan poikkeuksellisen herkkiä JAK-tyrosiinikinaasin ja BCL-perheen solukuolemaa säätelevien proteiinien estäjille. JAK-estäjillä pyritään hiljentämään samaa JAK-STAT-signalointireittiä, josta löysimme geneettisiä muutoksia ANKL-potilailla. Myeloproliferatiivisten sairauksien ja nivelreuman hoidossa käytettävillä JAK-estäjillä voitaisiin mahdollisesti tehostaa NK-soluisten syöpien hoitoa hyödyntämällä kyseisen solutyypin voimakasta riippuvuutta JAK-STAT-signaloinnin aktiivisuudesta. Tutkimuksemme valottaa geenitason muutoksia aiemmin tautimekanismeiltaan tuntemattomassa ANKL:ssa. Lääkeherkkyysseulonnan avulla pystyimme tunnistamaan potentiaalisia lääkeaineita ajatellen hoitokokeiluja harvinaisessa ANKL:ssa, jossa kliinisiä lääketutkimuksia pystytään harvoin toteuttamaan

Bayesian multi-source regression and monocyte-associated gene expression predict BCL-2 inhibitor resistance in acute myeloid leukemia

The FDA recently approved eight targeted therapies for acute myeloid leukemia (AML), including the BCL-2 inhibitor venetoclax. Maximizing efficacy of these treatments requires refining patient selection. To this end, we analyzed two recent AML studies profiling the gene expression and ex vivo drug response of primary patient samples. We find that ex vivo samples often exhibit a general sensitivity to (any) drug exposure, independent of drug target. We observe that this "general response across drugs" (GRD) is associated with FLT3-ITD mutations, clinical response to standard induction chemotherapy, and overall survival. Further, incorporating GRD into expression-based regression models trained on one of the studies improved their performance in predicting ex vivo response in the second study, thus signifying its relevance to precision oncology efforts. We find that venetoclax response is independent of GRD but instead show that it is linked to expression of monocyte-associated genes by developing and applying a multi-source Bayesian regression approach. The method shares information across studies to robustly identify biomarkers of drug response and is broadly applicable in integrative analyses

Targeting Key Survival Signaling Pathways for the Treatment of Leukemia

Leukemia is a cancer of the blood cells that has traditionally been treated with unselective chemotherapeutic agents. Advances in molecular biology and sequencing technologies have revolutionized the understanding of molecular and genetic factors in leukemia pathogenesis. Each leukemia patient harbors a unique set of genetic abnormalities, which result in impaired regulation of cell proliferation and differentiation. Increased cell proliferation is commonly mediated through overactive signaling cascades, such as JAK/STAT, PI3K/AKT/mTOR and MAPK pathways. The improved understanding of the molecular pathobiology has led to the development of small molecule inhibitors that can directly bind to target proteins and inhibit aberrant signaling. However, the molecular landscape of rare types of leukemia has not been comprehensively studied and our understanding of the relationship between cancer genotype, phenotype and drug function is limited. In study I, we aimed to identify novel driver mutations in large granular lymphocyte (LGL) leukemia. Earlier, our research group discovered that 40% of LGL leukemia patients had activating point mutations in the STAT3 gene. By using exome and targeted sequencing we identified 4/211 patients to carry an activating STAT5B mutation in the SH2 domain (N642H and Y640F). In study II, we identified STAT5B mutations in 6/68 (8%) T-cell acute lymphoblastic leukemia (T-ALL) patients. The finding represents a novel mechanism leading to the activation of the IL7R/JAK/STAT5 pathway. Our initial results also suggest that hyperactive STAT5B might be involved in the overexpression of anti-apoptotic BCL-xL. In study III, using a functional reporter assay we screened 306 oncology compounds to find potential hits that can decrease the cellular activity of mutant STAT3. The most potent targeted compounds inhibiting STAT3 activity were cyclin-dependent kinase (CDK), mammalian target of rapamycin (mTOR), heat shock protein 90 (Hsp90), and Janus kinase (JAK) inhibitors. Among these compounds only the Hsp90 inhibitors effectively inhibited both the mutant and wild type STAT3 phosphorylation and activity. In study IV, we developed a flow cytometry-based drug screening assay to assess cell population specific drug responses in heterogenous AML samples. Using the assay, we were able to simultaneously measure the drug responses of leukemic blasts, more mature leukemic cells and lymphocytes. The data showed that targeted therapies have different efficacies towards leukemic AML cells at distinct stages of myeloid differentiation. Particularly, BCL2 inhibitor venetoclax was toxic to immature blasts but was not effective against more differentiated monocytic and granulopoietic cells. In conclusion, we identified novel STAT5B mutations in two rare lymphoproliferative diseases, T-ALL and LGL leukemia. Furthermore, we demonstrate that our ex vivo flow cytometry-based drug screening platform can increase the functional understanding of drug effects.Leukemia on joukko erilaisia verisyöpiä, joiden pääasiallisena hoitomuotona käytetään jakautuviin syöpäsoluihin kohdistuvia solunsalpaajia. Viime vuosikymmeninä kehittyneet molekyylibiologiset menetelmät (kuten koko genomin sekvensointi) ovat lisänneet ymmärrystä leukemian syntymekanismeista. Tautigeenien tunnistaminen on johtanut satojen erilaisten syöpälääkkeiden kehittämiseen, jotka täsmällisesti hiljentävät tietyn syöpägeenin tuottaman proteiinin toimintaa ja täten syövälle tärkeiden signalointireittien aktiivisuutta. Väitöskirjatutkimuksen tavoitteena oli tunnistaa uusia patogeenisiä mutaatioita harvinaisissa T-solu leukemioissa ja tutkia miten uudet kohdennetut syöpälääkkeet tehoavat syöpäsoluja vastaan. Osatöissä 1. ja 2. löysimme aktivoivia, ennen tunnistamattomia STAT5B mutaatioita kahdesta eri leukemian alatyypistä (LGL-leukemia 2% potilasta, T-ALL 8% potilaista). STAT proteiinit ovat transkriptiofaktoreita, jotka säätelevät muiden geenien toimintaa ja yliaktiivisina estävät solujen normaalin ohjelmoidun kuoleman. Tulokset vahvistavat STAT3 ja STAT5B transkriptiofaktoreiden merkitystä T-soluisten leukemioiden synnyssä. Aikaisemmin tutkimusryhmämme havaitsi, että STAT3 on mutatoitunut 40%:lla LGL leukemia potilaista. Osatyössä 3. tutkimme in vitro -lääkeherkkyysanalyysin avulla 306 lääkkeen vaikutusta STAT3 transkriptiofaktorin aktiivisuuteen lymfoproliferatiivisissa taudeissa. CDK-, mTOR-, Hsp90- ja JAK-proteiineja estävät täsmälääkkeet osoittautuivat tehokkaimmiksi STAT3 aktiivisuuden hiljentäjiksi. Tulokset voivat olla hyödyllisiä suunniteltaessa uusia hoitoja STAT3 mutatoituneille leukemiapotilaille. Osatyössä 4. kehitettiin moderni virtaussytometriapohjainen lääkeherkkyysmenetelmä, jonka avulla arvioitiin eri solutyyppien lääkeherkkyyttä AML potilaiden heterogeenisistä luuydinnäytteistä. Tutkimuksemme osoitti, että täsmälääkkeillä on erilainen teho eri kypsyysvaiheessa olevia leukemiasoluja kohtaan. Erityisesti BCL2 estäjä venetoklaksi oli tehokkaampi, kun syöpäsolukko oli lähtöisin epäkypsemmistä myeloisista soluista (blasteista), kun taas teho laski syöpäsolukon kypsyessä monosyytti- tai granulosyytti-suuntaan. Tulos on ajankohtainen, sillä kliinisissä kokeissa venetoklaksi on antanut lupaavia tuloksia AML:n hoidossa. Tällä hetkellä on kuitenkin epäselvää, ketkä potilaista saavat hyvän hoitovasteen ja tuloksemme voivat auttaa näiden potilaiden tunnistamisessa. Tarkempi laboratorio-olosuhteissa suoritettu lääkeherkkyystutkimus voi myös auttaa löytämään juuri kyseiselle täsmälääkkeelle herkät potilaat

Functional Evaluation of Novel STAT3 Mutations Identified in Large Granular Lymphocytic Leukemia

Recently, our research group together with the Hematology Research Unit Helsinki, found that 31 of 77 patients (40%) with T-cell large granular lymphocytic leukemia have somatic point mutations in the Src Homology 2 (SH2) domain of the STAT3 gene. LGL leukemia is a rare and indolent disease characterized by the clonal expansion of large granular lymphocytes of unknown etiology. The aim of this master's thesis study was to elucidate whether the identified Y640F and D661V mutations can cause hyperactivity of the STAT3 protein and excessive proliferation of T-cells. STAT3 is a transcription factor that is known to have a key role in cell proliferation and apoptosis, and which is activated by the phosphorylation of receptor-associated kinases. The phosphorylation of tyrosine residue 705 in STAT3 induces dimerization and localization of the STAT3 dimer to the nucleus. In several cancers STAT3 protein has been reported to be constitutively active. Furthermore, previously published data strongly support our hypothesis that the mutations identified in LGL patients might cause STAT3 to be hyperactive resulting in inhibition of apoptotic pathways in cytotoxic T cells. In order to evaluate the function of the novel mutations, expression constructs of STAT3 containing the D661V and Y640F mutations were generated. In addition, lentiviral vectors were produced to establish a T-cell line (Jurkat) with stable expression of mutant STAT3. After the successful generation of STAT3 constructs and cell line models, several functional assays were performed. Transcriptional activity of STAT3 was measured by luciferase reporter assay and immunocytochemistry was used to determine whether the mutations promote nuclear localization of STAT3. STAT3 phosphorylation was examined by immunoblotting. In addition, quantitative RT-PCR was used to detect differential expression of five STAT3 target genes from patient samples. The results, particularly the luciferase reporter assay, indicated a significant difference between the mutant and wild type STAT3 providing strong evidence that the STAT3 mutants are transcriptionally more active. The localization assay, imaged by fluorescence microscopy, showed more STAT3 D661V and Y640F protein present in the nucleus when compared to wild type STAT3. However, the proliferation rate of mutant STAT3 expressing Jurkat cells was not increased. In addition, STAT3 target gene expression levels of two patient samples did not show large differences when compared to healthy LGL cells. As a result of these findings, it can be strongly hypothesized that aberrant STAT3 signaling underlies the cause of T-cell LGL leukemia. Understanding the molecular basis of LGL leukemia is important in order to develop diagnostic and therapeutic strategies for patients suffering from the disease. Since constitutively active STAT3 is common amongst many cancers and autoimmune disorders, activating mutations could possibly be found in these diseases. More careful sequencing studies of STAT3 upstream molecules are warranted as well, and will be performed in the future from leukemic LGL samples