Novel Genomic Biomarkers for Childhood Acute Lymphoblastic Leukemia

Akuutti lymfoblastileukemia (ALL) on lapsuusiän yleisin syöpä. Se voidaan luokitella B- ja T-solulinjojen taudeiksi taudin alkuperän mukaan. Näistä kahdesta leukemiatyypistä B-ALL on selkeästi yleisempi, ja kaikista lasten ALL:ista noin 85 % on B-solulinjan tauteja. ALL:n ennuste on nykyään erinomainen vuosikymmenien saatossa asteittain kehitettyjen lääkehoitojen ansiosta, ja niinpä B-ALL potilaista noin 90 % paranee pysyvästi. Taudin uusiutuessa ennuste on selvästi heikompi, ja erityisesti T-ALL on harvemmin parannettavissa uusiutumisen jälkeen. B-ALL voidaan luokitella useaan eri alatyyppiin erityisten geneettisten muutosten perusteella, ja tyypillisen tällaisen alaryhmän muodostavat potilaat, joiden leukemiasoluissa todetaan ETV6::RUNX1-fuusiogeeni. T-ALL:n luokittelua ei ole samalla tavalla vakiinnutettu ja niinpä tutkimukset, joissa haetaan ennusteellisia merkkiaineita, ovat hyvin ajankohtaisia. Tässä väitöskirjassa löydettiin kaksi uutta merkkiainetta T-ALL:aan hyödyntämällä aikaisemmin kerättyä laajaa verisairauksien geeni-ilmentymisen tietopankkia. PRMT7- ja SIX6-geenit ilmentyivät vahvasti T-ALL:ssa verrattuna muihin verisyöpiin ja terveisiin T-soluihin. Solukokeissa havainnoimme, että arginiinimetyylitransferaasi PRMT7 lisäsi T-ALL-solujen pesäkekasvua, muutti T- ALL:n patogeneesiin osallistuvien geenien ilmentymistä ja häiritsi RUNX1- proteiinin arginiinijäänteiden monometylaatiota ja sen toimintaa. Korkea PRMT7- ja SIX6-geenien ilmentyminen liittyi huonompaan ennusteeseen. T-ALL:ssa hyvin tunnettu transkriptiotekijä TAL1 sääteli suoraan SIX6:n ilmentymistä. Noin neljännes lapsuusiän ALL-tapauksista luokitellaan ETV6::RUNX1-alatyypin leukemiaksi. Tämän leukemiatyypin ennuste on yleensä erittäin hyvä, mutta tuntemattomasta syystä pieni osa potilaista reagoi huonosti alkuvaiheen lääkehoidolle, ja heillä on lisääntynyt taudin uusiutumisen riski. Tutkimushypoteesinamme oli, että hoitovasteen puutteen takana ovat geneettiset tekijät, ja tutkimme asiaa 175 potilaan näytteistä. Tunnistimme isoja geenialueiden puutoksia ja monistumia, jotka liittyivät hoitovasteeseen. Nopeasti hoitoon vastaavien leukemiasolujen geeneissä oli usein havaittavissa APOBEC- mutaatiojälki ja kromosomialue 12p puutos. Solut olivat myös usein aktiivisesti solusyklin jakautumisvaiheessa. Hitaasti hoitoon vastaavilla leukemiasoluilla havaittiin immunoglobuliinigeenin (kappa) uudelleenjärjestymiä ja kappa-deleetio- alueen muutoksia. Tästä johtuen solujen fenotyyppi muuttui, mikä viittaa B- solureseptorin signaloinnin aktivoitumiseen. Yhteenvetona voidaan todeta, että tässä väitöskirjatutkimuksessa tunnistettiin kaksi uutta merkkiainetta T-soluiseen ALL:aan sekä geneettisiä muutoksia, jotka selittävät hoitovastetta ETV6::RUNX1-tyypin leukemiassa. Tämän kaltaiset tutkimukset mahdollistavat tulevaisuudessa paremmin kohdennettujen hoitojen suunnittelun leukemiaan.Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. It can be classified into T and B cell lineage leukemias, with B-ALL being more common, and accounting for approximately 85% of cases. The survival rate of ALL patients has exceeded 90% due to significant improvements in treatment over the past decades. However, the prognosis of relapsed disease is much worse, and relapsed T-ALL is particularly difficult to cure. B-ALL can be divided into several subtypes based on the presence of specific genetic alterations. One of these is the ETV6::RUNX1 subtype, which is found in one-quarter of pediatric ALL cases. In contrast to B-ALL, T-ALL lacks a similar prognostic classification system. Therefore, studies seeking to identify clinically applicable biomarkers for T-ALL are highly relevant. In this thesis, we used a large gene expression data set of hematological malignancies and healthy samples to screen for potential candidate genes that are biologically relevant to T-ALL pathogenesis and could serve as prognostic biomarkers. Two genes, protein arginine methyltransferase 7 (PRMT7) and SIX homeobox 6 (SIX6), were highly expressed in the T-ALL samples. PRMT7 promoted the growth of T-ALL cells in colony formation assays, regulated the expression of various genes associated with T-ALL pathogenesis, and altered the arginine monomethylation of the RUNX1 transcription factor, leading to the altered expression of RUNX1 target genes. High PRMT7 expression was associated with a trend toward inferior event-free survival and overall survival. Similarly, high levels of SIX6 messenger RNA were associated with inferior event-free survival and overall survival. The expression of SIX6 was directly upregulated by TAL1, a master regulator of transcription in T-ALL. Patients with the ETV6::RUNX1 fusion typically have excellent outcomes. However, a subset of patients has a slow early response to therapy, which is associated with an increased risk of relapse. We hypothesized that secondary genetic alterations would determine the treatment response in ETV6::RUNX1-positive leukemia. In leukemia samples from a cohort of 175 pediatric ETV6::RUNX1 patients, we identified large genomic deletions and amplifications associated with early therapy response. The fast-responding leukemic blasts frequently exhibited the APOBEC mutational signature, had higher cell cycle activity, and were often missing a region of chromosome 12p. In contrast, blasts from slow-responding leukemias had frequent rearrangements in the immunoglobulin kappa gene and kappa-deleting elements. Upon treatment, the blasts with productive rearrangements were enriched, suggesting the activation of BCR signaling. In summary, we discovered two novel prognostic biomarkers for T-ALL and the response-associated genetic features of B-ALL. These findings may pave the way for more individualized therapy in pediatric ALL in the future

Activation of Intracellular Signalling Pathways in Adipose Stem Cell- mediated Immunosuppression

Background and aims: Adipose tissue is a source of multipotent adipose stromal/stem cells (ASCs). ASCs have excellent proliferation and multilineage differentiation capacity, low immunogenicity and promising immunosuppressive capacity. The immunosuppressive properties of ASCs have been previously studied but detailed intracellular mechanisms are still unknown. The aim of this study was to investigate the suppressive potential of ASCs in direct versus indirect co-culture with peripheral blood mononuclear cells (PBMCs) and to identify the signaling pathways (STAT3, STAT1, NF-κB and Smad1/5) that are activated during ASC-mediated immunosuppression. Methods: Four different ASC donors were used to study the immunosuppressive capacity of ASCs. Mixed lymphocyte reactions (MLR) using direct and indirect co-cultures of ASCs and PBMCs were used to study the immunosuppressive capacity of ASCs. Immunosuppression was analyzed using BrdU-ELISA. Activation of intracellular signaling pathways were analyzed using Western Blot analysis from direct and indirect MLR samples. Results: Strong immunosuppression on PBMC proliferation was obtained with three ASC donors. ASCs in direct co-culture with PBMCs had stronger immunosuppressive capacity compared to indirect co-cultures. ASCs cultured with MLR2 combination possessed stronger immunosuppressive capacity compared to MLR1 combination. There was variation in the activation of intracellular signaling pathways between different donors. Two signaling pathways (NF-κB and Smad1/5) were activated only in direct reactions compared to pathways STAT3 and STAT1, which were activated also in indirect reactions. NF-κB phosphorylation was inhibited in ASCs in direct reactions. Activation of Smad1/5 was donor-specific and it was activated with other ASC donor and inhibited with another ASC donor. In direct reactions STAT3 phosphorylation was inhibited and in indirect reactions STAT3 was mainly activated. STAT1 was phosphorylated in PBMCs with three ASCs donors and in two ASC donors in indirect reactions. Conclusion: ASCs have immunosuppressive capacity when co-cultured with PBMCs in both direct and indirect cultures. More studies with more ASC and PBMC donors are needed to obtain more detailed information about intracellular signaling behind the immunosuppression of ASCs

Omiikka työkaluksi leukemian hoitojen suunnitteluun

Kansallisia hankkeita genomitiedon tuomiseksi syöpädiagnostiikkaan on käynnissä useassa Euroopan maassa. Sekvensointiteknologian avulla voidaan samanaikaisesti tunnistaa yhden emäsparin mutaatioita, laajempia DNA:n lisäyksiä tai poistumia, translokaatioita sekä monimutkaisia kromosomitason muutoksia. Vain osa näistä muutoksista kartoitetaan nykyisillä diagnostisilla testeillä. RNA- tai epigeneettisillä profiileilla on osoitettu olevan diagnostista lisäarvoa. Tieto geenivirheistä yhdistettynä niiden luennan aktiivisuuteen mahdollistaa viallisten prosessien tunnistamisen. Merkittävä haaste on tuoda nämä tulokset hoitoa suunnittelevan asiantuntijatiimin käyttöön hoidon eri vaiheissa. Tarkastelemme lasten akuutin leukemian näkökulmasta syövän kartoitusta omiikan menetelmin sekä tämän siirtymistä kliiniseen käyttöön kansallisten ja kansainvälisten hankkeiden myötä.publishedVersionPeer reviewe

Arginine Methyltransferase PRMT7 Deregulates Expression of RUNX1 Target Genes in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with no well-established prognostic biomarkers. We examined the expression of protein arginine methyltransferases across hematological malignancies and discovered high levels of PRMT7 mRNA in T-ALL, particularly in the mature subtypes of T-ALL. The genetic deletion of PRMT7 by CRISPR-Cas9 reduced the colony formation of T-ALL cells and changed arginine monomethylation patterns in protein complexes associated with the RNA and DNA processing and the T-ALL pathogenesis. Among them was RUNX1, whose target gene expression was consequently deregulated. These results suggest that PRMT7 plays an active role in the pathogenesis of T-ALL.</p

Arginine Methyltransferase PRMT7 Deregulates Expression of RUNX1 Target Genes in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with no well-established prognostic biomarkers. We examined the expression of protein arginine methyltransferases across hematological malignancies and discovered high levels of PRMT7 mRNA in T-ALL, particularly in the mature subtypes of T-ALL. The genetic deletion of PRMT7 by CRISPR-Cas9 reduced the colony formation of T-ALL cells and changed arginine monomethylation patterns in protein complexes associated with the RNA and DNA processing and the T-ALL pathogenesis. Among them was RUNX1, whose target gene expression was consequently deregulated. These results suggest that PRMT7 plays an active role in the pathogenesis of T-ALL.publishedVersionPeer reviewe

Single cell characterization of B-lymphoid differentiation and leukemic cell states during chemotherapy in ETV6-RUNX1-positive pediatric leukemia identifies drug-targetable transcription factor activities

Background Tight regulatory loops orchestrate commitment to B cell fate within bone marrow. Genetic lesions in this gene regulatory network underlie the emergence of the most common childhood cancer, acute lymphoblastic leukemia (ALL). The initial genetic hits, including the common translocation that fuses ETV6 and RUNX1 genes, lead to arrested cell differentiation. Here, we aimed to characterize transcription factor activities along the B-lineage differentiation trajectory as a reference to characterize the aberrant cell states present in leukemic bone marrow, and to identify those transcription factors that maintain cancer-specific cell states for more precise therapeutic intervention. Methods We compared normal B-lineage differentiation and in vivo leukemic cell states using single cell RNA-sequencing (scRNA-seq) and several complementary genomics profiles. Based on statistical tools for scRNA-seq, we benchmarked a workflow to resolve transcription factor activities and gene expression distribution changes in healthy bone marrow lymphoid cell states. We compared these to ALL bone marrow at diagnosis and in vivo during chemotherapy, focusing on leukemias carrying the ETV6-RUNX1 fusion. Results We show that lymphoid cell transcription factor activities uncovered from bone marrow scRNA-seq have high correspondence with independent ATAC- and ChIP-seq data. Using this comprehensive reference for regulatory factors coordinating B-lineage differentiation, our analysis of ETV6-RUNX1-positive ALL cases revealed elevated activity of multiple ETS-transcription factors in leukemic cells states, including the leukemia genome-wide association study hit ELK3. The accompanying gene expression changes associated with natural killer cell inactivation and depletion in the leukemic immune microenvironment. Moreover, our results suggest that the abundance of G1 cell cycle state at diagnosis and lack of differentiation-associated regulatory network changes during induction chemotherapy represent features of chemoresistance. To target the leukemic regulatory program and thereby overcome treatment resistance, we show that inhibition of ETS-transcription factors reduced cell viability and resolved pathways contributing to this using scRNA-seq. Conclusions Our data provide a detailed picture of the transcription factor activities characterizing both normal B-lineage differentiation and those acquired in leukemic bone marrow and provide a rational basis for new treatment strategies targeting the immune microenvironment and the active regulatory network in leukemia

Therapeutic targeting of LCK tyrosine kinase and mTOR signaling in T-cell acute lymphoblastic leukemia

Relapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.Peer reviewe

Developmental partitioning of SYK and ZAP70 prevents autoimmunity and cancer

Even though SYK and ZAP70 kinases share high sequence homology and serve analogous functions, their expression in B and T cells is strictly segregated throughout evolution. Here, we identified aberrant ZAP70 expression as a common feature in a broad range of B cell malignancies. We validated SYK as the kinase that sets the thresholds for negative selection of autoreactive and premalignant clones. When aberrantly expressed in B cells, ZAP70 competes with SYK at the BCR signalosome and redirects SYK from negative selection to tonic PI3K signaling, thereby promoting B cell survival. In genetic mouse models for B-ALL and B-CLL, conditional expression of Zap70 accelerated disease onset, while genetic deletion impaired malignant transformation. Inducible activation of Zap70 during B cell development compromised negative selection of autoreactive B cells, resulting in pervasive autoantibody production. Strict segregation of the two kinases is critical for normal B cell selection and represents a central safeguard against the development of autoimmune disease and B cell malignancies.acceptedVersionPeer reviewe

Rare and low-frequency coding variants alter human adult height

Height is a highly heritable, classic polygenic trait with ~700 common associated variants identified so far through genome - wide association studies . Here , we report 83 height - associated coding variants with lower minor allele frequenc ies ( range of 0.1 - 4.8% ) and effects of up to 2 16 cm /allele ( e.g. in IHH , STC2 , AR and CRISPLD2 ) , >10 times the average effect of common variants . In functional follow - up studies, rare height - increasing alleles of STC2 (+1 - 2 cm/allele) compromise d proteolytic inhibition of PAPP - A and increased cleavage of IGFBP - 4 in vitro , resulting in higher bioavailability of insulin - like growth factors . The se 83 height - associated variants overlap genes mutated in monogenic growth disorders and highlight new biological candidates ( e.g. ADAMTS3, IL11RA, NOX4 ) and pathways ( e.g . proteoglycan/ glycosaminoglycan synthesis ) involved in growth . Our results demonstrate that sufficiently large sample sizes can uncover rare and low - frequency variants of moderate to large effect associated with polygenic human phenotypes , and that these variants implicate relevant genes and pathways