FLT3-ITD allelic ratio and HLF expression predict FLT3 inhibitor efficacy in adult AML

FLT3 internal tandem duplication (FLT3-ITD) is a frequent mutation in acute myeloid leukemia (AML) and remains a strong prognostic factor due to high rate of disease recurrence. Several FLT3-targeted agents have been developed, but determinants of variable responses to these agents remain understudied. Here, we investigated the role FLT3-ITD allelic ratio (ITD-AR), ITD length, and associated gene expression signatures on FLT3 inhibitor response in adult AML. We performed fragment analysis, ex vivo drug testing, and next generation sequencing (RNA, exome) to 119 samples from 87 AML patients and 13 healthy bone marrow controls. We found that ex vivo response to FLT3 inhibitors is significantly associated with ITD-AR, but not with ITD length. Interestingly, we found that the HLF gene is overexpressed in FLT3-ITD+ AML and associated with ITD-AR. The retrospective analysis of AML patients treated with FLT3 inhibitor sorafenib showed that patients with high HLF expression and ITD-AR had better clinical response to therapy compared to those with low ITD-AR and HLF expression. Thus, our findings suggest that FLT3 ITD-AR together with increased HLF expression play a role in variable FLT3 inhibitor responses observed in FLT3-ITD+ AML patients.Peer reviewe

KIT pathway upregulation predicts dasatinib efficacy in acute myeloid leukemia

Peer reviewe

Intrinsic resistance to PIM kinase inhibition in AML through p38α-mediated feedback activation of mTOR signaling

Although conventional therapies for acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL) are effective in inducing remission, many patients relapse upon treatment. Hence, there is an urgent need for novel therapies. PIM kinases are often overexpressed in AML and DLBCL and are therefore an attractive therapeutic target. However, in vitro experiments have demonstrated that intrinsic resistance to PIM inhibition is common. It is therefore likely that only a minority of patients will benefit from single agent PIM inhibitor treatment. In this study, we performed an shRNA-based genetic screen to identify kinases whose suppression is synergistic with PIM inhibition. Here, we report that suppression of p38α (MAPK14) is synthetic lethal with the PIM kinase inhibitor AZD1208. PIM inhibition elevates reactive oxygen species (ROS) levels, which subsequently activates p38α and downstream AKT/mTOR signaling. We found that p38α inhibitors sensitize hematological tumor cell lines to AZD1208 treatment in vitro and in vivo. These results were validated in ex vivo patient-derived AML cells. Our findings provide mechanistic and translational evidence supporting the rationale to test a combination of p38α and PIM inhibitors in clinical trials for AML and DLBCL

Quantitative scoring of differential drug sensitivity for individually optimized anticancer therapies

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

Hemap: An nteractive online resource for characterizing molecular phenotypes across hematologic malignancies

Large collections of genome-wide data can facilitate the characterization of disease states and subtypes, permitting pan-cancer analysis of molecular phenotypes and evaluation of disease contexts for new therapeutic approaches. We analyzed 9,544 transcriptomes from over 30 hematologic malignancies, normal blood cell types and cell lines, and show that the disease types can be stratified in a data-driven manner. We utilized the obtained molecular clustering for discovery of cluster-specific pathway activity, new biomarkers and in silico drug target prioritization through integration with drug target databases. Using known vulnerabilities and available drug screens in benchmarking, we highlight the importance of integrating the molecular phenotype context and drug target expression for in silico prediction of drug responsiveness. Our analysis implicates BCL2 expression level as important indicator of venetoclax responsiveness and provides a rationale for its targeting in specific leukemia subtypes and multiple myeloma, links several polycomb group proteins that could be targeted by small molecules (SFMBT1, CBX7 and EZH1) with CLL, and supports CDK6 as disease-specific target in AML. Through integration with proteomics data, we characterized target protein expression for pre-B leukemia immunotherapy candidates, including DPEP1. These molecular data can be explored using our freely available interactive resource, Hemap, for expediting therapeutic innovations in hematologic malignancies

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

Toimiva molekyylitarkkuuslääke akuutin myelooisen leukemian hoitoon

Acute myeloid leukemia (AML) is an aggressive and heterogeneous malignancy of the hematopoietic system. This thesis explores functional precision medicine approaches to target subsets of AML, in vitro, ex vivo and in the clinic. The experimental strategies focused on high-throughput drug sensitivity and resistance testing (DSRT), exome and RNA-sequencing. The real-time implementation of molecular and functional data for relapsed and refractory patients highlighted the opportunities of future individually tailored therapies in AML. We investigated the functional and molecular basis of cytarabine resistance to identify the molecular mechanisms of resistance and drugs that overcome the resistance. Cytarabine resistant AML cell lines MOLM-13 and SHI-1 variants were generated by long-term culturing. The cells were subjected to genomic sequencing, gene expression profiling and DSRT along with AML patient samples. We found a marked increase in glucocorticoid sensitivity in SHI-1 cytarabine resistant variants and in two AML patients treated with cytarabine-based regimens. Analysis of a larger set of relapsed or refractory AML patient samples revealed wild type FLT3 was significantly associated with sensitivity to glucocorticoids. The finding suggests glucocorticoids could be an effective class of drugs for a subset of cytarabine resistant AML patients with wild type FLT3 gene. We sought to identify drugs showing the discrepancy in responses between cell lines and patient samples, and potential drug sensitivity biomarkers. The drug responses, exome and RNA sequencing data were analyzed from 45 AML patient samples and 28 AML cell lines. Dasatinib, a multi tyrosine kinase inhibitor, was observed as one of the drugs with remarkably high sensitivity in the patient samples compared to the established cell lines. The upregulation of the KIT pathway was significantly associated with ex vivo dasatinib efficacy. The KIT pathway upregulation and ex vivo dasatinib sensitivity were found to be correlated with clinical response to dasatinib in three AML patients. Our results warrant testing dasatinib in a clinical trial for the AML patients with ex vivo dasatinib sensitivity and gene expression-based KIT pathway upregulation. A real-time precision systems medicine strategy was applied to identify targeted therapeutic options and biomarker associations in 252 samples from 186 AML patients. The integration of molecular profiling and DSRT data revealed ex vivo drug efficacies and underlying mutations and gene expression biomarkers for the subsets of AML patient samples. We developed a multidisciplinary functional precision medicine tumor board (FPMTB) to guide clinical therapy decisions for chemorefractory and recurrent (R/R) AML patients in a prospective real-time non-randomized clinical study. Our data suggest that functional precision medicine could have clinical utility, although it needs supportive evidence from formal prospective clinical trials. This thesis highlights novel avenues to define therapeutic opportunities for specific molecular subgroups of adult AML. The strategies described here could be useful for prioritizing drug development, identifying molecular subsets and drug response biomarkers, defining ideas for clinical trials, as well as the direct implementation of the therapies in individual AML patients in the FPMTB system.Akuutti myelooinen leukemia (AML) on hematopoieettisen järjestelmän aggressiivinen ja heterogeeninen pahanlaatuisuus. Tämä opinnäytetyö tutkii toiminnallisia tarkkuuslääketieteellisiä lähestymistapoja AML: n kohdealaryhmiin in vitro, ex vivo ja klinikalla. Kokeelliset strategiat keskittyivät suuritehoisiin lääkeherkkyyteen ja resistenssitestaukseen (DSRT), eksomiin ja RNA-sekvensointiin. Molekyyli- ja funktionaalisen datan reaaliaikainen käyttöönotto uusiutuneille ja tulenkestäville potilaille korosti mahdollisuuksia tulevaisuudessa yksilöllisesti räätälöityihin AML-hoitoihin. Tutkimme sytarabiiniresistenssin toiminnallista ja molekyylipohjaa tunnistaaksesi resistenssin molekyylimekanismit ja lääkkeet, jotka voittavat resistenssin. Sytarabiiniresistentit AML-solulinjat MOLM-13- ja SHI-1-variantit tuotettiin pitkäaikaisella viljelyllä. Soluille suoritettiin genomisekvensointi, geeniekspressioprofilointi ja DSRT yhdessä AML-potilasnäytteiden kanssa. Löysimme huomattavan lisääntyneen glukokortikoidiherkkyyden SHI-1-sytarabiiniresistenteissä muunnoksissa ja kahdessa AML-potilaassa, joita hoidettiin sytarabiinipohjaisilla hoito-ohjelmilla. Suuremman uusiutuneiden tai tulenkestävien AML-potilasnäytteiden analyysi paljasti villityypin FLT3: n liittyneen merkittävästi herkkyyteen glukokortikoideille. Tulos viittaa siihen, että glukokortikoidit voivat olla tehokas lääkeryhmä sytarabiiniresistenttien AML-potilaiden alaryhmälle, jolla on villityypin FLT3-geeni. Etsimme lääkkeitä, jotka osoittavat eroja solulinjojen ja potilasnäytteiden vasteissa ja potentiaalisissa lääkeherkkyysbiomerkkeissä. Lääkevasteet, exome- ja RNA-sekvensointitiedot analysoitiin 45 AML-potilasnäytteestä ja 28 AML-solulinjasta. Dasatinibi, monityrosiinikinaasin estäjä, havaittiin yhtenä lääkkeistä, joilla oli huomattavan suuri herkkyys potilasnäytteissä verrattuna vakiintuneisiin solulinjoihin. KIT-reitin säätäminen liittyi merkittävästi dasatinibin tehoon ex vivo. KIT-reitin säätelyn ja ex vivo dasatinibiherkkyyden havaittiin korreloivan dasatinibin kliinisen vasteen kanssa kolmella AML-potilaalla. Tuloksemme edellyttävät dasatinibin testaamista kliinisessä tutkimuksessa AML-potilaille, joilla on ex vivo dasatinibiherkkyys ja geeniekspressiopohjainen KIT-reitin säätely. Reaaliaikaista tarkkuussysteemilääketieteen strategiaa sovellettiin kohdennettujen hoitovaihtoehtojen ja biomarkkeriyhdistysten tunnistamiseksi 252 näytteessä 186 AML-potilaasta. Molekyyliprofilointi- ja DSRT-tietojen integrointi paljasti ex vivo lääketehokkuudet ja taustalla olevat mutaatiot sekä geeniekspressiobiomarkkerit AML-potilasnäytteiden osajoukoille. Kehitimme monitieteisen toiminnallisen tarkkuuslääketieteen kasvainkortin (FPMTB) ohjaamaan kemorefraktiivisten ja toistuvien (R / R) AML-potilaiden kliinisiä hoitopäätöksiä prospektiivisessa reaaliaikaisessa ei-satunnaistetussa kliinisessä tutkimuksessa. Tietojemme mukaan funktionaalisella tarkkuuslääkkeellä voi olla kliininen hyöty, vaikka se tarvitsee tukevaa näyttöä virallisista prospektiivisista kliinisistä tutkimuksista. Tämä opinnäytetyö tuo esiin uusia keinoja määritellä terapeuttiset mahdollisuudet aikuisten AML: n spesifisille molekyylialaryhmille. Tässä kuvatut strategiat voivat olla hyödyllisiä lääkekehityksen priorisoimiseksi, molekyyliosien ja lääkevasteen biomarkkereiden tunnistamiseksi, kliinisten tutkimusten ideoiden määrittelemiseksi sekä terapioiden suoraksi toteuttamiseksi yksittäisissä AML-potilaissa FPMTB-järjestelmässä