Dasatinib and Navitoclax Act Synergistically to Target NUP98-NSD1 +/FLT3-ITD+ Acute Myeloid Leukemia

Leukemia https://doi.org/10.1038/s41375-018-0327-2

ARTICLE

Acute myeloid leukemia Dasatinib and navitoclax act synergistically to target NUP98-NSD1 +/FLT3-ITD+ acute myeloid leukemia

1 2 1 3,4 3 Jarno L. Kivioja ● Angeliki Thanasopoulou ● Ashwini Kumar ● Mika Kontro ● Bhagwan Yadav ● 1 1 1 2 3,4 Muntasir M. Majumder ● Komal K. Javarappa ● Samuli Eldfors ● Juerg Schwaller ● Kimmo Porkka ● Caroline A. Heckman1

Received: 28 June 2018 / Revised: 21 September 2018 / Accepted: 10 October 2018 © Springer Nature Limited 2018

Abstract Acute myeloid leukemia (AML) with co-occurring NUP98-NSD1 and FLT3-ITD is associated with unfavorable prognosis and represents a particularly challenging treatment group. To identify novel effective therapies for this AML subtype, we screened patient cells and engineered cell models with over 300 compounds. We found that mouse hematopoietic progenitors co-expressing NUP98-NSD1 and FLT3-ITD had signiﬁcantly increased sensitivity to FLT3 and MEK-inhibitors compared to cells expressing either aberration alone (P<0.001). The cells expressing NUP98-NSD1 alone had signiﬁcantly P = NUP98-NSD1+/FLT3- +

1234567890();,: 1234567890();,: increased sensitivity to BCL2-inhibitors ( 0.029). Furthermore, ITD patient cells were also very sensitive to BCL2-inhibitor navitoclax, although the highest select sensitivity was found to SRC/ABL-inhibitor dasatinib + (mean IC50 = 2.2 nM). Topoisomerase inhibitor mitoxantrone was the least effective drug against NUP98-NSD1 /FLT3-ITD + AML cells. Of the 25 significant hits, four remained significant also compared to NUP98-NSD1-/FLT3-ITD+ AML patients. We found that SRC/ABL-inhibitor dasatinib is highly synergistic with BCL2-inhibitor navitoclax in NUP98-NSD1 +/FLT3-ITD+ cells. Gene expression analysis supported the potential relevance of dasatinib and navitoclax by revealing significantly higher expression of BCL2A1, FGR, and LCK in NUP98-NSD1+/FLT3-ITD+ patients compared to healthy CD34+ cells. Our data suggest that dasatinib–navitoclax combination may offer a clinically relevant treatment strategy for AML with NUP98-NSD1 and concomitant FLT3-ITD.

Introduction recurrent fusion gene involving nucleoporin-98-kDa (NUP98) at 11p15.5 was first described more than two Acute myeloid leukemia (AML) is a complex disease decades ago and since then at least 32 distinct NUP98 characterized by various chromosomal alterations [1]. A fusion partner genes have been identified [2–6]. Regardless of the fusion partner, disruption of NUP98 leads to changes in the stoichiometry and nucleo-cytoplasmic transport functions of the nuclear pore complex [7]. While NUP98 Supplementary information The online version of this article (https:// rearrangements form the second largest gene fusion network doi.org/10.1038/s41375-018-0327-2) contains supplementary fi material, which is available to authorized users. in AML, they also de ne a biologically distinct subgroup of patients with poor prognosis [8–10]. In this study, we * Caroline A. Heckman focused on the t(5;11)(q35;p15.4) translocation [11, 12], fi caroline.heckman@helsinki. which is the most common NUP98 rearrangement in cyto- 1 Institute for Molecular Medicine Finland, Helsinki Institute of Life genetically normal AML. According to Hollink et al. [13], Science, University of Helsinki, Helsinki, Finland the resulting NUP98-NSD1 is present in 16% of pediatric 2 Department of Biomedicine, University Children’s Hospital, and 2% of adult cytogenetically normal AML patients with University of Basel, Basel, Switzerland over 80% also carrying activating FLT3-ITD (internal tan- NUP98- 3 Hematology Research Unit Helsinki, University of Helsinki, dem duplication) mutations. Co-occurrence of Helsinki, Finland NSD1 and FLT3-ITD is associated with especially low – 4 Department of Hematology, Helsinki University Hospital complete remission and survival rates in AML [13 17]. Comprehensive Cancer Center, Helsinki, Finland During a three-year follow-up, the event-free survival rate J. L. Kivioja et al. among NUP98-NSD1+/FLT3-ITD+ patients was shown to epigenetic modifier genes IDH1/2, NPM1,orMLL-rear- be 14% [14]. Although, allogeneic hematopoietic stem cell rangements. BM mononuclear cells (MNCs) were enriched transplantation may improve survival [14–16], no evidence by gradient centrifugation (Ficoll-Paque Premium™,GE regarding the efficacy of specific therapeutic agents exists Healthcare, Little Chalfont, United Kingdom) and either for these patients. cryopreserved until analysis or immediately suspended in The main objective of the present study was to discover Mononuclear Cell Medium (MCM; PromoCell, Heidelberg, novel candidate drugs and drug combinations for NUP98- Germany) supplemented with 0.5 µg/ml gentamicin and 2.5 NSD1+/FLT3-ITD+ AML patients and to understand the µg/ml amphotericin B. From AML patients with multiple contribution of NUP98-NSD1 to AML drug response. We samples, sample with the highest leukemic burden was utilized a standardized high-throughput drug sensitivity and included. Germline controls (skin biopsies) were collected resistance testing assay [18–20] for analyzing hematopoietic from two of the NUP98-NSD1+/FLT3-ITD+ patients for mouse cell lines transduced with NUP98-NSD1 and FLT3- exome sequencing. Table 1 describes clinical and pheno- ITD retroviruses independently, or co-transduced with both. typic features of the NUP98-NSD1+/FLT3-ITD+ study Results from the experimental cell lines were supported by patients (data lock 7 September 2018). similar drug sensitivity assessment of bone marrow (BM) cells from NUP98-NSD1+/FLT3-ITD+ and NUP98-NSD1-/ Mouse BM-derived cells FLT3-ITD+ AML patients. RNA sequencing was performed on experimental mouse cells and patient cells to Lineage marker-depleted BM cells from BALB/c mice were understand gene expression changes induced by NUP98- transduced with empty vector, NUP98-NSD1, and FLT3- NSD1 and FLT3-ITD that may associate with the drug ITD encoding retroviruses alone or co-transduced with responses. NUP98-NSD1 and FLT3-ITD as previously described [17]. We found that NUP98-NSD1+/FLT3-ITD+ patient cells The (“preleukemic”) cells were expanded in RPMI-1640 exhibit significantly high sensitivity to multi-target tyrosine (Lonza, Basel, Switzerland) complete medium (10% fetal kinase inhibitor (TKI) dasatinib and pan-BCL2 inhibitor bovine serum, 1% L-glutamine, and 1% penicillin- navitoclax. Genes encoding dasatinib target proteins LCK streptomycin (10,000 U/ml each) supplemented with and FGR, and navitoclax target BCL2A1 were significantly interleukin-6 (10 ng/ml), mouse interleukin-3 (6 ng/ml), and upregulated in NUP98-NSD1+/FLT3-ITD+ AML cells mouse stem cell factor (10 ng/ml) (Peprotech, Rocky Hill, relative to healthy CD34+ cells. By screening experimental NJ, USA), or propagated in vivo (“leukemic”)byBM mouse cell lines, we observed that co-expression of NUP98- reconstitution in irradiated syngeneic recipient mice as NSD1 and FLT3-ITD induces a significant increase in previously described [17]. Additional cell lines included Ba/ selective FLT3 and MEK inhibitor responses compared to F3 cells (ATCC CRL-12015) transduced with lentiviruses cells expressing FLT3-ITD alone. We also found that carrying either empty backbone vector (pLeGO-iCer2) or dasatinib and navitoclax are highly synergistic in leukemic pLeGO-iCer2-NUP98-NSD1 (Supplementary Figure 1). NUP98-NSD1+/FLT3-ITD+ human and mouse cells, sug- The lentiviral packaging, transduction, and cell selection gesting targeted therapies may be available for this clini- methods are described in the Supplementary information cally challenging subtype of AML. file.

Compound collection Materials and methods The compound collection included 306 drugs including Patient material approved drugs (U.S. Food and Drug Administration or European Medicines Agency), investigational compounds The study was approved by the local institutional review and probes (Supplementary Table 1). The compounds were board at the Helsinki University Hospital (permit numbers reformatted in 100% dimethyl sulfoxide or water and plated 239/13/03/00/2010, 303/13/03/01/2011, Helsinki Uni- in ﬁve concentrations over a 10,000-fold range (e.g., 1– versity Hospital Ethics Committee). The clinical samples 10,000 nM) on 384-well ﬂat clear bottom white were collected after informed consent and in accordance polystyrene tissue culture treated microplates (Corning, with the Declaration of Helsinki. BM aspirates were New York, NY, USA) using an Echo 550 acoustic collected from all available Finnish NUP98-NSD1+/FLT3- dispenser (Labcyte Inc, Sunnyvale, CA, USA). The ITD+ AML patients. Control samples were collected from microplates were kept in pressurized StoragePods® (Roylan ten healthy donors and nine NUP98-NSD1-/FLT3-ITD+ Developments Ltd., Fetcham, United Kingdom) under inert AML patients, which lacked concurrent mutations in nitrogen gas until use. Dasatinib and navitoclax act synergistically to target. . .

Drug sensitivity and resistance testing , + The plated compounds were resuspended by adding 5 µl of ﬂ

weak (36%), culture medium to each well and brie y shaking the plates

+ with a Titramax 1000 platform shaker (Heidolph, Schwa- bach, Germany). Afterwards, 20 µl of cell suspension – , CD19-, CD7-, CD9-, (2000 10,000 cells/well) was added to the wells with a , CD15; a proportion of cells + (70%), CD4 + Multidrop™ Combi Reagent Dispenser (Thermo Fisher + ] , (weak 24%), CD3-, CD15 + 58 ﬁ

, MPO Scienti c, Carlsbad, CA, USA). The plates were incubated , CD4 + +

, CD25 for 72 h in a humidiﬁed incubator at +37°C. Cell viabilities + , CD4-, CD3-, CD13- (26%), CD41a, CD42a&61-, CD42b-, TdT + , CD135 were measured before and after the incubation using Cell- + + , CD64

+ Titer-Glo® reagent according to the manufacturer’s instructions (Promega, Madison, WI, USA). The lumines- , CD117 (21%) (26%), HLA-DR , CD14 ® + +

+ cence from each well was captured with a PHERAstar FS + , CD19-, CD7 + , CD133-, CD2 microplate reader (BMG Labtech, Offenburg, Germany) + , CD22 , CD65 and normalized to negative (dimethyl sulfoxide) (n = 16) , + + , MPO , HLA-DR + , CD56-, CD203c, CD123 World Health Organization [ n = + + and positive (100 µmol/L benzethonium chloride) ( 8) , CD36 + in gDNA calculated by comparing peak areas generated with +

] ’

, CD15 control wells. The threshold for plate-wise Z values was + 57 WHO

, HLA-DR – FLT3 , CD11b weak (16%), CD11b 0.5 1.0 [21]. The dose-response curves were generated in + , HLA-DR + + (28%), MPO sex,

+ Dotmatics Browser v4.8 (Dotmatics Ltd., Herts, United + S Kingdom) and ﬁtted through a four parametric logistic ﬁt , CD13 , CD13 , CD11b + + +

, CD117 function that considers top and bottom asymptote, slope, + and the inﬂection point. To improve the interpretation of weak (66%), CD135 , CD38 , CD79-, TdT , CD38 , CD13 + + + +

+ drug-sample interactions, dose-response curves were con- -ITD and wild type

overall survival, verted to a drug sensitivity score (DSS) as described [22]. , IREM2-, CD14-, CD64-, CD71 (48%), CD33 , CD38 , CD33 , CD65 , CD33 + +

FLT3 DSS is a normalized area under the curve, calculated rela- OS + + + + + tive to total area between 10 and 100% inhibition. The (16%), CD96 CD105 + CD34 BM immunophenotype CD177 CD36 cyCD79a-, NG2-, cyCD3-, CD16-, CD117 male, selective drug sensitivity score (sDSS) was calculated for

M each drug by subtracting the median DSS of control sample a = 5.4 8.1 CD33 from the DSS of experimental sample (sDSS DSSsample- (months) 53.3* CD34 12.1 CD34

female, DSScontrol). Pilot combinatorial screens were performed by F treating experimental cell lines with 70 drugs ± dasatinib

. Allelic Ratio between (100 nM). The comprehensive follow-up screening was patients A.R

+ performed using 8 × 8 or 6 × 6 drug concentration matrices. The zero-interaction potency (ZIP) synergy scores (δ) were calculated in SynergyFinder [23, 24]. Allo-HSCT (planned) Therapy OS inhibitor allo-HSCT, azacitidine, DLI allo-HSCT, re-induction

NUP98-NSD1 Gene expression proﬁling donor leukocyte infusion, FLT3- ITD A. R.

DLI Paired-end RNA sequencing (RNA-Seq) was performed on total RNA (1–3 µg) from experimental BALB/c cell lines, BM MNCs of three AML patients with NUP98-NSD1 +/FLT3- + NUP98 NSD1-/FLT3- +

FLT3-ITD length (bp) ITD , nine - ITD AML patients, and BM CD34+ cells from four healthy donors. The CD34+ cells were enriched with the EasySep® Human + 70 57 0.346 Induction, consolidation, BM blast count (%) 75 57 0.182 Induction x2, TKI, MTOR 40 213 0.310 Induction, consolidation, 95 57 0.403 Induction, consolidation, patients belong to adverse cytogenetic risk group based on European LeukemiaNet (2017) criteria [ CD34 Selection Kit (StemCell Technologies, Vancouver,

+ Canada). RNA extractions were done using the RNeasy

allogeneic hematopoietic stem cell transplantation, Mini Kit or miRNeasy Kit (Qiagen, Hilden, Germany), cation ﬁ while RNA quality was assessed with the 2100 Bioanalyzer AML with maturation WHO classi Acute monocytic leukemia AML with maturation Acute monocytic leukemia using the Agilent RNA 6000 or Pico Kit (Agilent Tech- NUP98-NSD1 Clinical and phenotypic characteristics of the 32 A 54 39 58 allo-HSCT nologies, Santa Clara, CA, USA). RNA concentrations were measured with the Qubit ﬂuorometer (Thermo Fisher Sci- age,

Patient remains in complete remission at data lock ﬁ 7499 F/ fragment analysis (supplemental information), A Table 1 Patient S/ All four a 600 M/ 3600 F/ 3660 M/ enti c). Subsequently, RNA was depleted of ribosomal J. L. Kivioja et al.

RNAs (Ribo-Zero rRNA Removal Kit; Epicentre, Madison, NSD1+ BALB/c and Ba/F3 cells (mean sDSS = 17.5). WI, USA), purified (RNeasy® MinElute® Cleanup Kit, Interestingly, the leukemic NUP98-NSD1+/FLT3-ITD+ Qiagen), and reverse transcribed to double-stranded cDNA cells were resistant (sDSS < 0) to BCL2 inhibitors (navito- with the Superscript® Double-Stranded cDNA Synthesis Kit clax; sDSS = −7.9, obatoclax; sDSS = −6.0). Although the (Thermo Fisher Scientific) using random hexamers (New mechanism of resistance remains unclear, it may arise from England BioLabs, Ipswich, MA, USA). RNA-Seq libraries the ability of FLT3-ITD to up-regulate MCL1 through its were prepared with Nextera (Epicentre), and ScriptSeq STAT5-docking domains [29]. In contrast to BCL2 inhi- technologies (Illumina, San Diego, CA, USA), bar-coded, bitor resistance, the dual positive cells had significantly (P and enriched by ligation PCR according to the manu- < 0.01) increased selective sensitivity to FLT3 and MEK facturer’s instructions (Illumina). PCR fragments were size inhibitors (Fig. 1b), with quizartinib (mean sDSS = 22.8) selected (350–700 bp) from a 2% agarose gel, purified with and pimasertib (mean sDSS = 13.2) being the most effec- the QIAquick Gel Extraction Kit (Qiagen), and sequenced tive FLT3 and MEK inhibitors (Fig. 1c). High MEK inhi- with an Illumina HiSeq™2000 or HiSeq™2500 instrument. bitor sensitivity in the NUP98-NSD1+/FLT3-ITD+ cells Sequenced reads were aligned against human and mouse may result from co-activation of STAT5 and MAPK path- (BALB/c) reference genomes. The spliced alignment of the way downstream of FLT3 signaling as reported [30]. processed paired-end reads from human samples was per- Consistent with earlier studies and drug testing results, our formed against the reference genome (Ensembl GRCh38) phospho-flow analysis showed increased phosphorylation with guidance of the Ensembl reference gene models of STAT5 and ERK1/2 in NUP98-NSD1+/FLT3-ITD+ (Ensembl v82) with the gap-aware STAR aligner [25]. Raw BALB/c BM cells compared to cells expressing either of the read counts were normalized by trimmed mean of M values two aberrations alone (Supplementary Figure 2). The drug scaling normalization method and log2 CPM (counts per screening data of all experimental mouse cell lines are million) values calculated with the edgeR R package shown in Supplementary Table 2 and Supplementary (3.18.1) [26]. The RNA-Seq data preprocessing and ana- Figure 3. lysis pipeline have been previously described [27, 28]. Dasatinib sensitivity is highly specific for NUP98- Statistical analysis NSD1+/FLT3-ITD+ AML cells

Statistical analyses were performed in GraphPad Prism In addition to the mouse cell lines, we evaluated drug v6.01 (GraphPad Software, La Jolla, CA, USA). DSS sensitivity profiles of BM MNCs from four NUP98-NSD1 comparisons were computed with the Mann–Whitney U test +/FLT3-ITD+ AML patients and ten healthy donors (Fig. 2 and correlation analyses with the Spearman’s rank correla- and Supplementary Table 3). Of the 171 drugs tested in all tion coefficient test. Gene expression values were compared samples, 99 (57.9%) had a higher median DSS in the using the unpaired two-sample t tests. All statistical tests NUP98-NSD1+/FLT3-ITD+ BM MNCs compared to heal- applied in this study were two-tailed and P values below thy BM MNCs indicating select targeting of the leukemic 0.05 were considered significant. cell, 51 (30%) had no response (DSS = 0) in either group, and 21 (12.9%) had a higher median DSS in the healthy BM MNCs (Supplementary Figure 4). We identified 25 statisti- Results cally significant differences between the NUP98-NSD1 +/FLT3-ITD+ AML patients and healthy controls (Table 2). Molecular driven responses to BCL2, FLT3, and MEK The most selective drugs included kinase inhibitors, BCL2 inhibitors in experimental mouse cell lines inhibitor navitoclax, four HDAC inhibitors, and three heat shock protein 90 (HSP90) inhibitors. Of the top candidate To discover small-molecule inhibitors with activity against drugs, SRC-family protein kinase inhibitor dasatinib had the NUP98-NSD1, we tested lineage marker-depleted BALB/c lowest mean half maximal inhibitory concentration (IC50 = BM-derived cells expressing NUP98-NSD1 and FLT3-ITD 2.2 nM, 95% CI -1.4–5.8 nM). The topoisomerase II inhi- alone or both together as well as NUP98-NSD1+ Ba/F3 bitor mitoxantrone (P < 0.01) was the least effective drug cells with 306 drugs (Fig. 1a). By comparing the selective against the NUP98-NSD1+/FLT3-ITD+ AML patient cells drug sensitivities (sDSS) between cell lines, we discovered (Table 2). Two of the patients (600 and 3660) had pre- that NUP98-NSD1+ BALB/c BM cells have significantly viously been exposed to a combination of cytarabine and (P = 0.029) higher select sensitivity to BCL2 inhibitors idarubicin, thus the tested cells were likely representative of compared to leukemic cells co-expressing NUP98-NSD1 a drug resistant clone that had been selected for by that and FLT3-ITD (Fig. 1b). Navitoclax was identified as the treatment. Nevertheless, treatment naïve cells from patient most selective BCL2 inhibitor (n = 4) towards NUP98- 3600 and 7499 showed comparable resistance to Dasatinib and navitoclax act synergistically to target. . .

Fig. 1 Drug testing results from experimental mouse cell lines. At the inhibitors in each experimental mouse cell line. Significant differences hit identification stage, each experimental cell line was tested once between the cell lines are denoted with an asterisk (*P = 0.029; ****P with a library of 306 compounds. a The histograms show the 25 most < 0.0001). c The lower panel shows dose-response curves of the most effective drugs in Ba/F3 and BALB/c BM-derived cells expressing selective BCL2 inhibitor (navitoclax) in the NUP98-NSD1+ BALB/c NUP98-NSD1 [1, 2], FLT3-ITD [3], or both aberrations together cells, and the most selective FLT3 (quizartinib) and MEK inhibitor [4, 5]. The preleukemic cells were cultured in vitro while the leukemic (pimasertib) in the dual positive BALB/c cells. Drug concentrations cells were propagated in vivo. b The box plots show the individual are shown on the x-axis and inhibition percentage on the y-axis data points and mean sDSS of several BCL2, FLT3, and MEK topoisomerase II inhibitors suggesting that NUP98-NSD1 tivozanib (P = 0.03) had significantly higher DSS in +/FLT3-ITD+ AML cells may be intrinsically resistant to NUP98-NSD1+/FLT3-ITD+ cells compared to NUP98- this class of drugs. Additionally, cells from the index patient NSD1-/FLT3-ITD+ cells, while mitoxantrone (P = 0.007) (600) showed high resistance to nucleoside analogues such had significantly lower DSS (Fig. 3). This provides addi- as clofarabine, cladribine, and fludarabine. tional support that topoisomerase II inhibitor resistance may To evaluate the differential responses further, we com- be specific for NUP98-NSD1+/FLT3-ITD+ AML. pared drug sensitivities between NUP98-NSD1+/FLT3-ITD As NUP98/11p15 translocations were recently shown to + and NUP98-NSD1-/FLT3-ITD+ AML patients. Of the 25 affect CD34+ hematopoietic precursor cells in myeloid drugs, belinostat (P = 0.02), dasatinib (P = 0.03), and neoplasms [31], we also assessed the impact of BM blast J. L. Kivioja et al.

Fig. 2 Drug testing results from patient cells. The waterfall plots show patient. BM aspirates from the patients were collected at relapse (R) or the 20 most and least selective drug responses in BM MNCs of four at diagnosis (D). The index patient’s (600) BM MNCs were tested NUP98-NSD1+/FLT3-ITD+ AML patients in comparison to healthy with 171 drugs, whereas the cells from patient 3600, 3660, and 7499 donor BM MNCs. The sDSS was calculated for each drug by sub- were tested with 306 drugs. Drugs denoted with an asterisk were not tracting the median DSS of ten healthy donors from the DSS of a tested in the index case count on DSS. A trend towards positive correlation was navitoclax (patient 600) (56% inhibition) or 30 nM navito- found for SRC/ABL inhibitor dasatinib (R = 0.800) indi- clax (patient 7499) (61.8% inhibition). Maximum inhibition cating that blast content may explain some of the variation (100%) was achieved at an area of ≥3 nM dasatinib com- in DSS between the NUP98-NSD1+/FLT3-ITD+ patients. bined with ≥1000 nM of navitoclax. In leukemic mouse In NUP98-NSD1-/FLT3-ITD+ BM MNCs the correlation cells, the highest synergy was achieved at 3 nM dasatinib coefficient was 0.25 (Supplementary Figure 5). combined with 300 nM navitoclax and maximum inhibition (74%) achieved at 10 nM dasatinib combined with 10,000 Dasatinib–navitoclax combination is synergistic in nM navitoclax. leukemic cells co-expressing NUP98-NSD1 and FLT3- ITD NUP98-NSD1+/FLT3-ITD+ BM MNCs show increased expression of LCK, FGR, and BCL2A1 compared to To identify drugs that could synergize with dasatinib for healthy CD34+ cells greater efficacy, we performed a pilot combinatorial screen with the mouse cell lines using 70 drugs ± dasatinib (100 NUP98-NSD1 has been reported to enforce the nM) (Fig. 4a). Interestingly, dasatinib had the strongest constitutive expression of distinct HOX-AB cluster genes impact on DSS in NUP98-NSD1+/FLT3-ITD+ mouse cells [9, 13, 16, 32, 33]. Considering this, we initially analyzed as indicated by the slope of linear regression line (Supple- the expression of several HOX-AB genes in patient cells and mentary Figure 6). In the dual positive cells, dasatinib experimental cell lines to validate our samples. Consistent significantly increased sensitivity to FLT3 and MEK inhi- with earlier studies, we observed high expression of HOX- bitors. Moreover, a modest increase in BCL2 inhibitor A9, HOX-A10, HOX-B3, -B4, -B5, -B6, and -B7 genes in the sensitivities was observed in the cells expressing NUP98- NUP98-NSD1+/FLT3-ITD+ BM MNCs compared to heal- NSD1 alone (Fig. 4 and Supplementary Table 4). To follow- thy CD34 + cells (Fig. 6a). In BALB/c BM-derived cells, up on these responses, we selected eleven drug pairs for NUP98-NSD1 alone induced expression of several HOX-AB more comprehensive evaluation using dose-response genes, which remained upregulated in the dual positive cells matrices (Supplementary Table 5-6). The combination of (Fig. 6b). dasatinib and navitoclax had the highest synergistic activity Subsequently, we analyzed the expression of dasatinib in patient cells (patient 600: δ = 14.0; patient 7499: δ = 6.1) and navitoclax target genes in primary patient cells and and leukemic BALB/c BM-derived cells (δ = 9.4) co- experimental cell lines (Fig. 6c–f). Intriguingly, we expressing NUP98-NSD1 and FLT3-ITD (Fig. 5). In observed a significant increase in expression of the dasatinib patient cells, the highest synergy was achieved at an area targets LCK (P = 0.005) and FGR (P < 0.001) and the where 0.1 nM dasatinib was combined with 300 nM navitoclax target BCL2A1 (P = 0.028) in NUP98-NSD1 Dasatinib and navitoclax act synergistically to target. . .

Table 2 Differential analysis of Drug name Drug target Phase of DSS median 95%CI DSS 95% CI DSS P value drug sensitivities in BM MNCs clinical difference (patients) (healthy) of NUP98-NSD1+/FLT3-ITD+ study patients (n = 4) compared to healthy donors (n = 10) (P < Sensitive AZD8055 mTOR Phase 1 20.54 (4.1, 38.9) (1.1, 5.6) 0.004 0.01) Refametinib MEK1/2 Phase 1/2 19.60 (8.3, 26.8) (0.0, 3.0) 0.002 Tivozanib FLT3, VEGFR1-3, Phase 2 18.66 (11.5, 28.9) (−0.3, 6.4) 0.001 c-Kit, PDGFR Idelalisib PI3K, p110 δ- Phase 3 17.35 (4.4, 32.5) (1.1, 3.9) 0.004 selective Pimasertib MEK1/2 Phase 2 17.12 (6.2, 24.7) (0.3, 3.2) 0.002 Dasatinib ABL1, c-KIT, SRC, Approved 16.27 (11.9, 23.2) (−0.2, 7.1) 0.003 LCK BIIB021 HSP90 Phase 2 16.22 (21.5, 27.8) (7.0, 9.8) 0.002 Navitoclax BCL2, BCL-xL, Phase 2 12.37 (12.1, 41.1) (9.6, 12.8) 0.002 BCL-w, BCL2A1, MCL1 Lestaurtinib FLT3, JAK2, TrkA- Phase 3 11.56 (9.6, 20.4) (3.0, 6.6) 0.002 C Doramapimod pan-p38 MAPK, c- Phase 1/2 11.26 (4.9, 19.9) (0.0, 3.6) 0.001 RAF, FYN, LCK Foretinib MET, VEGFR2 Phase 2 10.83 (3.1, 25.8) (2.9, 5.0) 0.004 AZD7762 Chk1 Probe 10.27 (7.5, 24.3) (5.3, 8.3) 0.008 Luminespib HSP90 Phase 2 10.19 (21.4, 26.7) (11.5, 15.8) 0.002 OSI-027 mTOR Phase 1 9.94 (6.7, 18.7) (0.6, 4.2) 0.002 Linsitinib IGF1R, IR Phase 2 9.47 (3.2, 17.0) (0.6, 3.3) 0.004 Tanespimycin HSP90 Phase 2 9.19 (12.6, 24.3) (7.0, 9.6) 0.002 Everolimus mTOR Approved 8.23 (2.8, 14.0) (−0.5, 2.6) 0.002 Belinostat HDAC Phase 2 7.24 (20.9, 29.3) (15.3, 18.3) 0.002 Axitinib VEGFR, PDGFR, Approved 5.21 (2.6, 14.9) (1.6, 4.3) 0.004 KIT Danusertib Aurora kinase A-C, Phase 2 4.94 (10.6, 13.5) (4.7, 8.1) 0.002 Abl, c-RET, LCK Entinostat HDAC Phase 2 4.93 (2.5, 20.4) (3.9, 5.8) 0.004 Pictilisib PI3Kα/δ Phase 2 4.47 (8.9, 16.7) (6.5, 8.5) 0.002 Panobinostat HDAC Phase 3 4.20 (22.9, 30.3) (19.4, 22.6) 0.002 Vorinostat HDAC Approved 4.19 (9.0, 15.4) (5.8, 9.0) 0.002 Resistant Mitoxantrone Topoisomerase II Approved −6.27 (−2.0, 5.8) (5.9, 8.6) 0.008

+/FLT3-ITD+ BM MNCs compared to healthy CD34+ Discussion cells. Moreover, in the BALB/c BM cells expressing NUP98-NSD1 alone, BCL2A1 expression (5.4) was higher In this study, our objective was to identify novel candidate compared to BALB/c BM cells transduced with mock (0.0) drugs and drug combinations for the treatment of AML or FLT3-ITD alone (2.5). The BCL2 inhibitor resistant patients with concomitant NUP98-NSD1 and FLT3-ITD NUP98-NSD1+/FLT3-ITD+ BALB/c cells had the highest alterations by high-throughput drug screening and mole- MCL1 expression and the lowest BCL2 expression com- cular profiling. NUP98-NSD1+/FLT3-ITD+ AML patients pared to BCL2 inhibitor sensitive NUP98-NSD1+ BALB/c are a high-risk patient group that typically have reduced cells. This finding may suggest that co-expression of event-free survival, often develop chemo refractory disease, NUP98-NSD1 and FLT3-ITD leads to BCL2 inhibitor and are in need of novel treatment options. Here, we pro- resistance via upregulation of MCL1 and repression of vide evidence that NUP98-NSD1+/FLT3-ITD+ BM MNCs BCL2. Additionally, to investigate protein level expression have significantly increased select sensitivity to multi- of SRC-family kinases in NUP98-NSD1+/FLT3-ITD+ cells, kinase inhibitor dasatinib and BCL2 inhibitor navitoclax we analyzed phosphokinase antibody array results pre- and that these cells are resistant to topoisomerase II inhi- viously performed on the index patient (600) [18]. We bitors. We demonstrate that combined treatment of these found that FYN and LCK had increased phosphorylation cells with dasatinib and navitoclax is highly synergistic. Our compared to healthy controls in all three samples collected data suggest that increased expression of LCK, FGR, and from the index patient at different stages of disease pro- BCL2A1 may play important roles in sensitizing NUP98- gression (Supplementary Figure 7). NSD1+/FLT3-ITD+ AML cells to SRC/ABL inhibitor J. L. Kivioja et al.

identification of histone methyltransferase and chromatin co-factor inhibitors [33, 36, 37]. Deshpande et al. [36] found that DOT1L inhibitor EPZ004777 leads to increased differentiation and apoptosis of NUP98-NSD1 transformed murine hematopoietic cells, while another study reported that histone methyltransferase inhibitor suramin likely inhibits NSD1 [38]. The potential benefits of incorporating CREB-binding protein (CBP)/p300, FLT3 and HDAC inhibitors into the treatment algorithm for NUP98-NSD1+ patients have also been discussed [9, 13, 14, 39]. Our compound collection included only one CBP/p300 inhibitor (C646), however, only experimental cell lines were tested with this compound and no marked effects were observed. As shown in Table 2, out of the six HDAC and six FLT3 inhibitors tested in all patient samples, four HDAC (belinostat, entinostat, vorinostat, panobinostat) and two FLT3 inhibitors (lestaurtinib, tivozanib) were among the top candidate drugs. Based on these findings, our data suggest that future research should explore the potential efficacy of HDAC and FLT3 inhibitors in NUP98-NSD1+/FLT3-ITD+ AML cells. Earlier studies showed that NUP98-NSD1 defines a relatively rare, yet clinically significant subset of AML patients with unfavorable prognosis [13, 14, 40]. Due to infrequency of these patients, sample size was a major limiting factor affecting our study. To compensate for this limitation and achieve high accuracy, two control groups NUP98-NSD1 Fig. 3 Comparison of drug responses between NUP98-NSD1+/− (healthy donors and negative AML patients) AML patients with FLT3-ITD. a The floating bars show the range and and experimental mouse cell lines were used. Another median DSS of the 25 most differentially sensitive drugs tested in BM limitation was that our three-day, cell viability-based, drug MNCs of four NUP98-NSD1+/FLT3-ITD+ AML patients (orange), - + screening assay was suboptimal for capturing histone nine NUP98-NSD1 /FLT3-ITD AML patients (dark blue), and ten fi healthy donors (light blue). Drug names are shown on the y-axis and methyltransferase inhibitor ef cacies, which often require DSS on the x-axis. Significant differences between the two AML 7–14 days of treatment for pronounced effects on cell dif- patient groups are denoted with an asterisk (*P < 0.05; **P < 0.01). ferentiation or proliferation [41, 42]. Graphs b, c show the dose-response curves of dasatinib and navitoclax Based on the frequent co-occurrence of NUP98-NSD1 in the three groups with mean and standard deviation for each FLT3- concentration and ITD [14, 17], we assessed how their co- expression impacts drug response. Although only one mouse model (retroviral expression in BM cells of BALB/c dasatinib and BCL2 inhibitor navitoclax induced cell death. mice) was used, our data shows that co-occurrence of the The potential relevance of LCK is strengthened by the two lesions drastically changes the drug sensitivity land- observation that three of the top candidate drugs (3/24), scape in BALB/c BM cells (Supplementary Figure 3A-E). namely dasatinib, doramapimod, and danusertib, exert We found that FLT3 and MEK inhibitor sensitivities are potency against LCK (Table 2). Consistent with our find- significantly higher in cells co-expressing NUP98-NSD1 ings, a previous study has shown that LCK is a primary and FLT3-ITD compared to cells expressing either of the target of dasatinib and a critical signaling molecule in two aberrations alone. We also observed that the dual NUP214-ABL1+ T-cell acute lymphoblastic leukemia with positive cells are resistant to BCL2 inhibitors in contrast to potential to induce rapid complete cytogenetic remission as the cells expressing NUP98-NSD1 that had significantly monotherapy [34, 35]. increased sensitivity to BCL2 inhibitors. The finding sug- The present study is the first cancer pharmacopeia-wide gests that NUP98-NSD1 may induce the expression of anti- drug screening study systematically assessing novel small- apoptotic BCL2 family proteins such as BCL2A1. molecule inhibitors for NUP98-NSD1+/FLT3-ITD+ AML Interestingly, BCL2 inhibitor venetoclax has shown patients. Due to involvement of histone methyltransferase promising results in clinical trials and in vitro studies among NSD1, earlier studies on this topic have merely focused on patients with mixed-lineage leukemia (MLL) fusions Dasatinib and navitoclax act synergistically to target. . .

Fig. 4 Pilot combinatorial drug screening. a The heatmap shows DSS classes described below the figure. Graphs b–d illustrate FLT3, MEK, of 70 drugs tested with and without additional dasatinib (100 nM) in and BCL2 inhibitor responses (DSS) in the presence and absence of experimental mouse cells. Numbers 1–7 on top of the graph refer to dasatinib. The floating bars show median DSS with range. Significant cell lines, while annotation colors on the y-axis indicate different drug differences are denoted with an asterisk (*P = 0.023; **P < 0.01) overexpressing homeobox A and B (HOX-AB) cluster genes explore whether aberrant histone methylation plays a role in [20, 43, 44]. Considering that MLL and NSD1 genes share sensitizing NUP98-NSD1+ BM cells to BCL2 inhibitors several common functional domains and that over- such as navitoclax. It also remains to be studied whether the expression of HOX-AB cluster genes is well-established results presented here may be applicable to patients with among NUP98-NSD1+ AML patients [9, 13, 16, 32, 33], functionally similar NUP98 rearrangements such as high BCL2 inhibitor sensitivity in NUP98-NSD1+ cells may NUP98-MLL or NUP98-KDM5A. result via a common mechanism. In MLL-AF4+acute lym- In clinical trials, BCL2 inhibitors and TKIs have pro- phoblastic leukemia, venetoclax sensitivity was shown to duced only moderate effects as single agents with responses result from the ability of MLL-AF4 to activate BCL2 usually being incomplete and transient [44, 47, 48]. To through aberrant H3K79 methylation activities [45], which address this challenge, we systematically evaluated pair- is also a critical factor for NUP98-NSD1-driven leukemic wise drug combinations for NUP98-NSD1+/FLT3-ITD+ transformation [36]. A recent study has shown that NUP98- AML patients using dose-response matrices [24]. In patient NSD1 physically interacts with MLL1 and the NSL histone cells and leukemic mouse cells co-expressing NUP98- modifying complexes [46]. Additional studies should NSD1 and FLT3-ITD, the dasatinib–navitoclax combination J. L. Kivioja et al.

Fig. 5 Dose-response synergy screen. The two-dimensional contour expressing NUP98-NSD1 or FLT3-ITD alone (e, f). The synergy δ- plots show areas of synergy (red) and antagonism (green) for eight scores were calculated based on % inhibition values using the ZIP- concentrations of dasatinib (y-axis) and navitoclax (x-axis) in the index method in SynergyFinder [23, 24]. The most synergistic areas are patient (a), patient 7499 (b), leukemic and preleukemic BALB/c BM indicated with a square inside of each plot cells co-expressing NUP98-NSD1 and FLT3-ITD (c, d), and in cells had the highest synergy of the tested combinations, while survival BCL2 family members upregulated by FLT3-ITD high synergies were also observed for a navitoclax–qui- [29, 53, 54]. zartinib combination (Supplementary Table 6). Synergistic In contrast to the BCL2 inhibitor sensitive patient cells, interactions between BCL2 inhibitors (ABT-737, veneto- the leukemic NUP98-NSD1+/FLT3-ITD+ mouse cells were clax, sabutoclax) and TKIs (dasatinib and sorafenib) have resistant (sDSS < 0) to BCL2 inhibitors suggesting that our been reported in myeloid and lymphoid malignancies con- mouse model does not fully recapitulate the molecular ﬁrming the potential importance of our ﬁndings [49–53]. In complexity of patient cells. At least two of the patients lymphoid neoplasms, the synergy between BCL2 inhibitors (patients 600 and 3660) carried hotspot mutations in the and SRC/ABL inhibitor dasatinib has been linked to the DNA-binding domain of Wilms’ tumor suppressor gene ability of dasatinib to increase the NOXA/MCL-1 ratio and WT1 (Supplementary Table 7-8), which may explain dis- decrease BCL2A1 levels [50], and to concomitant induction crepancies in BCL2 inhibitor sensitivities between the of BIM expression and inhibition of MCL-1 upregulation experimental NUP98-NSD1+/FLT3-ITD+ mouse cells and [51]. In chronic myeloid leukemia, BCL2 inhibitor sabu- the patient cells. Interestingly, wild type WT1 has been toclax was shown to sensitize quiescent blast crisis leu- shown to function as a direct transcriptional regulator of kemic stem cells to dasatinib mediated cell death by BCL2 [55] and loss of its ability to activate pro-apoptotic abrogating BM niche-dependent dasatinib resistance [52]. Bak due to a mutation has been associated with the devel- Furthermore, in hematopoietic cell lines ABT-737 has been opment of Wilms’ tumor [56]. More recent work has shown shown to restore FLT3 inhibitor sensitivity by targeting pro- that WT1 mutations associate with increased ex vivo Dasatinib and navitoclax act synergistically to target. . .

Fig. 6 Gene expression analysis. The heatmaps show unsupervised package pheatmap (1.0.8). c, d The box plots illustrate the range and hierarchical clustering of RNA sequenced human (a) and experimental mean expression of dasatinib and navitoclax target genes in the pri- mouse cells (b) based on the mean centered expression (log 2 CPM) of mary human cells, while histograms show the expression of corre- HOX-AB genes. The clustering was done using Euclidean distance sponding genes in experimental BALB/c BM cells (e, f). Signiﬁcant with complete linkage method. The genes are listed on the right side of differences are denoted with an asterisk (*P = 0.028; **P = 0.006; the heatmap and samples below. The heatmaps were generated by R ***P = 0.0006) sensitivity to BCL2 inhibitor venetoclax in AML [20]. NUP98-NSD1 and FLT3-ITD. Our data suggest that While up to 45% of NUP98-NSD1+ AML patients carry NUP98-NSD1+/FLT3-ITD+ AML patients may not beneﬁt WT1 mutations [13], future research should explore how from the use of topoisomerase II inhibitors as part of front- these mutations impact BCL2 inhibitor responses and line induction therapy. It remains to be studied whether BCL2-family gene expression in cells expressing NUP98- replacement of topoisomerase II inhibitors with a targeted NSD1 alone or in conjunction with FLT3-ITD. agent such as dasatinib could improve outcomes of NUP98- In summary, the experimental evidence presented here NSD1+ AML patients if it was added to the usual induction provides new insights into treatment optimization for therapy. NUP98-NSD1+ AML as well as proof-of-concept studies with results supporting clinical investigations of targeted Acknowledgements We thank the patients and healthy donors who therapies for high-risk AML patients with concomitant participated in this study. We would also like to thank staff at the J. L. Kivioja et al.

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