ALBERT

Beschreibung: Several promising new, targeted agents are being developed for the treatment of AML. The BH3 mimetic venetoclax (ABT-199) is a specific inhibitor of BCL2, with results from a phase 2 study showing transient activity of venetoclax in relapsed/refractory AML (Konopleva et al, 2014). The bone marrow (BM) microenvironment is known to protect AML cells from drug therapy and we showed earlier that conditioned medium (CM) from BM stromal cells applied to AML patient cells conferred resistance to venetoclax, which could be reversed by the addition of the JAK1/2 inhibitor ruxolitinib (Karjalainen et al, 2015). Here, we investigated the mechanisms mediating the BM stromal cell induced resistance to venetoclax and its reversal by ruxolitinib. To identify the soluble factor(s) contributing to stroma-induced protection of BCL2 inhibition, we analyzed the cytokine content of 1) CM from the human BM stromal cell line HS-5, 2) CM from BM mesenchymal stromal cells (MSCs) isolated from AML patients, 3) supernatants from BM aspirates collected from AML patients, and 4) supernatants from BM aspirates collected from healthy donors. Although expression levels varied, the cytokines detected were similar among the different samples. In HS-5 CM, IL-6, IL-8 and MIP-3α were among the most abundant cytokines. In addition, gene expression analysis showed the receptors for these cytokines were expressed in AML patient samples. IL-6, IL-8 and MIP-3α were added individually to mononuclear cells collected from AML patients, which were then treated with venetoclax. However, none of the cytokines alone could mimic the reduced sensitivity to venetoclax conferred by the HS-5 CM suggesting that stromal cell induced cytoprotection is likely multi-factorial. Next we tested the effect of AML-derived BM MSCs on the ex vivo response of AML patient samples (n=8) to ruxolitinib or venetoclax alone or in combination in a co-culture setting. Apoptosis assays showed negligible effects of ruxolitinib at a concentration of 300 nM, while venetoclax at a dose of 100 nM induced reduction in the percentage of CD34+ AML cells. Co-treatment with venetoclax and ruxolitinib demonstrated synergistic effects in 6 out of 8 samples and significantly reduced the number of CD34+ AML cells. Mechanistic studies showed that ruxolitinib treatment inhibited the BM stromal medium-induced expression of BCL-XL mRNA on AML cells and the drugs in combination down-regulated BCL2, MCL1 and BCL-XL protein expression, which was in correlation with sensitivity to the drugs. To further evaluate the ability of the venetoclax and ruxolitinib combination to eradicate leukemic cells in vivo we used an orthotopic xenograft model of AML. NSG mice were injected with genetically engineered MOLM-13luc cells and after engraftment treated with venetoclax (25 mg/kg, i.p.), ruxolitinib (50 mg/kg BID, p.o) or both and imaged once per week for 4 weeks. At the end of the treatment period bioluminescent imaging showed significantly reduced leukemia burden in the ruxolitinib and venetoclax co-treated mice compared to controls demonstrating superior anti-tumor efficacy than either agent alone (Figure 1). In summary, our data demonstrate that the combined blockade of JAK/STAT and BCL2 pathways with ruxolitinib and ventoclax is synergistic in ex vivo co-culture models and in vivo in an AML mouse model. The addition of ruxolitinib was able to overcome intrinsic resistance to venetoclax by reducing expression of MCL1, a known escape mechanism of BCL2 inhibition. These results support further clinical investigation of this combination, particularly for relapsed/refractory AML. Disclosures Porkka: Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Wennerberg:Pfizer: Research Funding. Gjertsen:BerGenBio AS: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Kinn Therapeutics AS: Equity Ownership. Heckman:Celgene: Research Funding; Pfizer: Research Funding.

Beschreibung: While the majority of acute myeloid leukemia (AML) patients respond to induction chemotherapy, disease recurrence and drug resistance is common. Recently, mutations underlying AML pathogenesis have been extensively characterized by sequencing large numbers of samples obtained at diagnosis. However, mutations driving disease progression and drug resistance in relapsed AML are not well characterized. In addition, understanding the clonal composition of relapsed AML is compounded by interference of donor cell variants present in those patients who have received an allogeneic hematopoietic stem cell transplant (alloHSCT). In this study we sought to identify mutations and copy number aberrations associated with development of drug resistant AML, and at the same time develop methods to identify and filter out donor variants. For the study we analyzed samples from patients who had relapsed after therapy (N=18) by exome sequencing. This included a set of patients where diagnosis and relapse samples were available (n=10), and one patient with diagnosis, remission and relapse samples. All patients had received prior chemotherapy and a subset had relapsed after receiving an allogeneic hematopoietic stem cell transplant (alloHSCT, n=6). Four patients had secondary AML that had developed after treatment for earlier hematologic malignancy. Tumor DNA was from bone marrow mononuclear cells and germline DNA from matched skin biopsies. Exome libraries were prepared then sequenced with the Illumina HiSeq instrument. Sequence data was processed and somatic variants identified as described previously (Koskela et al., NEJM, 2012). We identified relapse specific and relapse enriched somatic mutations by comparing mutation profiles of diagnosis and relapse samples. Donor derived germline variants in chimeric samples from patients relapsing after alloHSCT were identified with a bioinformatic methodology utilizing the dbSNP population variant database. Somatic mutations called from chimeric samples were filtered for common population variants present in the donor’s genome. Rare donor derived population variants that have not been previously described were identified as variants not present in the patient’s germline genome and which had similar tumor variant allele frequencies as the common donor derived variants. We estimated the level of chimerism based on the variant allele frequencies of all donor derived variants. In chimeric samples, the number of donor derived variants vastly exceeded the number of somatic mutations in AMLs (Fig 1). Donor cell content varied widely ranging from close to 100% in a post transplant remission sample to 10-40% in relapse samples. In post-transplant samples, we identified on average 6800 donor germline variants within the exome-capture regions, many of which occurred within cancer genes which could potentially be misinterpreted as driver mutations. Many recurrent driver mutations in cancer genes were identified in the relapse samples: FLT3 (n=6, 33%), DNMT3A (n=4, 22%), NPM1 (n=2, 11%), WT1 (n=2, 11%), TP53 (n=2, 11%), CBL (n=2, 11%), NRAS (n=1, 6%), KRAS (n=1, 6%), IDH1 (n=1, 6%), PHF6 (n=1, 6%) and PTPN11 (n=1, 6%). In several cases, we observed that relapse-specific driver mutations occurred in the same genes or pathways that already had initial mutations at diagnosis. For example, one patient’s AML had a FLT3-ITD at diagnosis; at relapse an activating mutation in CBL and a loss of function mutation in PTPN11 were acquired. Both CBL and PTPN11 act downstream of FLT3 (Fig 2). In two patients with a heterozygous WT1 mutation at diagnosis, we found additional WT1 mutations or deletion of the remaining wild type allele in the relapse sample, suggesting full loss of normal WT1 function contributes to disease progression. Our results suggest that AML progression and drug resistance may be caused by strengthening aberrant signaling through pathways already affected by a mutation present at diagnosis. Hence, the pattern of mutual exclusivity of mutations to genes affecting the same pathway, which has been observed in diagnostic samples, does not occur at relapse. On the contrary, in several cases the relapse specific mutations affected genes in pathways already affected at diagnosis. In addition, we show that donor derived germline variants can be identified and filtered from exome sequence data. Figure 1 Figure 1. Disclosures Porkka: BMS: Honoraria; BMS: Research Funding; Novartis: Honoraria; Novartis: Research Funding; Pfizer: Research Funding. Kallioniemi:Medisapiens: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Beschreibung: Background Ex vivo drug sensitivity testing of cancer cells taken directly from patients would significantly facilitate optimization of clinical therapies. However, in the past, such testing has been performed in suboptimal conditions, where patient cells gradually stop proliferating and undergo apoptosis, with poor translation of Results. More reliable prediction of drug sensitivity is needed and recent focus has been directed towards Methods that take into account the supporting impact of the surrounding tumor microenvironment. Primary leukemia cell viability and long-term survival ex vivo can be promoted with co-culture Methods using stromal cells (McMillin et al. 2013). While high throughput (HT) drug testing enables rapid assessment of sensitivity to 100s of drugs or drug combinations, application of co-culture Methods is challenging considering the mixed readouts from multiple cell types. In this study we describe a HT platform based on stroma-conditioned medium for assessing the anti-leukemic activity of compounds against fresh and vital biobanked primary leukemia samples ex vivo. Methods Stroma-conditioned medium (CM) was collected from the HS-5 human bone marrow (BM) cell line and combined with RPMI medium for drug sensitivity testing. Mononuclear cell medium (Promocell) was used as the standard medium comparison. Sensitivity of primary leukemia or healthy cells to 306 approved and investigational drugs was measured at 5 different concentrations covering a 10,000-fold concentration range. Cell viability was measured after 72 h with the CellTiter-Glo assay and dose response curves generated for each tested drug. Drug sensitivity scores (DSS) were calculated based on the area under the dose response curve. Here, we compared comprehensive drug sensitivity ex vivo responses between stroma-conditioned medium and standard medium using mononuclear cells from 8 acute myeloid leukemia (AML) patients and 4 healthy donors. Results HS-5 CM supported fresh and biobanked primary AML cells, promoting proliferation and overall survival. Freshly isolated AML cells had a mean viability of 123% after 3 days in CM compared to 59% in the absence of CM. The viability of biobanked cells was 85% with CM vs. 20% in conventional medium. Improved ex vivo cell survival increased the therapeutic window of drug sensitivity testing and more drugs could be assessed with CM compared to conventional medium. Results from different healthy samples tested with the same type of medium were highly similar, but sensitivities differed significantly when comparing CM to standard medium Results. In contrast, drug sensitivity Results of AML cells from different patients were more diverse, reflecting the heterogeneity of the disease. However, comparison of CM and standard medium drug sensitivities of cells from individual AML patients showed modest differences that were primarily indicative of the increased proliferation of cells incubated with CM. Overall, both AML and healthy cells showed greater sensitivity to anti-mitotic drugs when incubated with CM. For example, the average DSS of vinblastine for healthy controls was 17 in CM vs. 9 in standard medium. In addition, AML cells often exhibited increased sensitivity to JAK inhibitors such as ruxolitinib when tested with CM compared to standard medium (DSS 14 vs. 9). In contrast, stress-related protein-targeting drugs (e.g. HSP90 inhibitors) and certain tyrosine kinase inhibitors (e.g. dasatinib, quizartinib) exhibited reduced efficacy when AML cells were incubated with CM compared to conventional media. This may be due to soluble factors present in CM that mimic the protection provided by the BM niche. Conclusions Our data support the concept that conditioned medium from stromal cells improves application of drug sensitivity testing to AML patient samples ex vivo. Stromal medium supports both fresh and biobanked AML cells, likely providing environmental cues present in the BM niche and necessary for AML cell growth and survival. This may lead to more reliable ex vivo assessment of the anti-leukemic activity of compounds for cells from leukemia patients. Importantly, stromal cell based conditions support the growth of vital biobanked leukemia samples and enable use of retrospective samples for a multitude of assays including HT drug testing. Disclosures: Porkka: Novartis: Consultancy, Research Funding, Speakers Bureau; BMS: Consultancy, Research Funding, Speakers Bureau. Kallioniemi:Medisapiens: Membership on an entity’s Board of Directors or advisory committees; Roche: Research Funding.

Beschreibung: AML patients (pts) carrying the fms-related tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) have a poor prognosis. Combination of chemotherapy with tyrosine kinase inhibitors (TKIs) has improved survival of these pts, but a large proportion of them still die of their disease. Nucleoside analogs (NAs) are the backbone of several upfront and salvage chemotherapy regimens for AML, including FLT3-ITD. Although these agents have significant antileukemic activity, they are not effective in eradicating leukemia stem cells (LSCs), the likely reason for treatment failures in AML. Consequently, new strategies are needed to improve the outcome for this and other molecular subsets of AML pts. 8-Cl-Ado is a novel, ribose-containing, RNA-directed nucleoside analog which, different from other NAs, is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. Among the AML molecular subsets, we identified FLT3-ITD blasts as one of the most sensitive to 8-Cl-Ado; however the mechanism of this differential effect remains unknown. Ex-vivo treatment with 8-Cl-Ado induced dose-dependent growth inhibition and apoptosis in FLT3-ITD AML cell lines and primary blasts including the LSC-enriched CD34+CD38- immunophenotypic subpopulation, with IC50s ranging from 200 nM to 1400 nM and a negligible effect on normal hematopoietic stem cells. In an orthotopic murine model, mice xenografted with FLT3-ITD-positive MV4-11 cells and treated (upon engraftment) with 75 mg 8-Cl-Ado/kg/day through an implanted osmotic pump survived significantly longer than vehicle-treated controls (median survival 45 days vs. 27.3 days, p=0.002). MicroRNA-155 (miR-155) is the most over-expressed miRNA in FLT3-ITD and reportedly plays a key role in FLT3-ITD blast hyper-proliferation [PMID 20656931, PMID 25971362]. Thus, silencing of miR-155 has been proposed as a novel therapeutic approach for FLT3-ITD AML [PMID 25971362]. As 8-Cl-Ado is incorporated mainly into RNA, we reasoned that it could also be incorporated into miR-155 (and other miRNAs). Consistent with our hypothesis, we detected co-localization of 8-Cl-Ado and miR-155 in FLT3-ITD primary blast cells and MV4-11, using fluorescence-labeled 8-Cl-Ado (8-Cl-Ado-FAM) and miR-155 staining (SmartFlare probes), suggesting that 8-Cl-Ado interacts directly with miR-155. Using quantitative RT-PCR we demonstrated ~50% miR-155 down-regulation in 8-Cl-Ado-FAM or 8-Cl-Ado-treated MV4-11 cells and FLT3-ITD primary blast cells, compared to vehicle-treated controls. On a molecular level, 8-Cl-Ado-dependent miR-155 down-regulation was accompanied by up-regulation of SHIP1, a bona fide miR-155 target phosphatase that decreased p-AKT levels thereby negatively regulating FLT3-ITD-dependent AKT signaling required for leukemia cell growth and survival. This effect also disrupted the interaction of AKT and ErbB3 binding protein (Ebp1), a highly expressed protein that regulates p53 expression and prevents DNA fragmentation and apoptosis in normal and leukemic cells. Thus, we hypothesized by disrupting the AKT/Ebp1 interplay via miR-155 down-regulation, 8-Cl-Ado induces pro-apoptotic Ebp1-dependent p53 expression and activation and leukemia cell death. Indeed, we showed that 8-Cl-Ado treatment caused AKT/Ebp1 dissociation and p53 activation in primary FLT3-ITD AML blasts. Conversely, overexpression of miR-155 reversed 8-Cl-Ado-induced apoptosis. By combining 8-Cl-Ado with the TKI quizartinib we elicited a synergistic anti-leukemic effect in primary AML blasts [combination index (CI) values

Beschreibung: Key Points BM stroma-derived conditions protect AML patient cells against topoisomerase II and BCL2 inhibitors, as well as several classes of TKIs. JAK1/2 inhibitor ruxolitinib reverses cytoprotection against BCL2 antagonist venetoclax, suggesting a novel combinatorial treatment.

Beschreibung: Background T-cell acute lymphoblastic leukemia (T-ALL) is caused by the cooperation of multiple oncogenic lesions. Recent evidence supports that IL-7 and its receptor IL-7R contribute to T-ALL development (Zenatti et al, 2011). The two main pathways induced by IL-7R are JAK/STAT5 and PI3K/Akt/mTOR. Activating mutations to IL7R, JAK1, JAK2 or JAK3 are estimated to occur in 20-30% of all T-ALL patients (Cools 2013). STAT5 plays an important role in many hematologic malignancies but constitutive STAT5 activation often is a secondary event. Mutations in STAT5B (N642H) were recently described in LGL-leukemia in patients with an unusually aggressive and fatal form of the disease (Rajala et al, 2013). In other cancers, including ALL, patients with mutations in STAT5B have not been described. Here we report novel activating STAT5B mutations as drivers of T-ALL. Methods We performed exome sequencing of bone marrow (BM) samples from an 18-year-old female with relapsed T-ALL. Targeted next-generation amplicon sequencing and Sanger sequencing was used to analyze the region encoding the STAT5B SRC homology 2 (SH2) domain including the N642, T648 and I704 codons in a cohort of 17 adult and pediatric T-ALL patients treated at HUCH 2008-2013. For functional studies STAT5B expression vectors with the N642H, T648S or I704L mutation and an expression vector with both N642H and T648S mutations were used to transiently transfect HEK293 cells. To investigate the effect on transcriptional activity we co-transfected the mutant constructs with a STAT5 luciferase reporter plasmid and used Western blot analysis to study the phosphorylation status of the generated constructs. For drug sensitivity of STAT5B mutated cells we performed ex vivo drug testing on primary blasts from the index patient using a comprehensive set of 202 oncology drugs (approved and in clinical development). Each drug was tested over a 10,000-fold concentration range. Results Sequencing of the index patient revealed 3 different somatic missense mutations in STAT5B (T648S, N642H, I704L) and mutations in KRAS, WT1 and SUZ12. No mutations affecting the JAK genes or IL7R were detected. All STAT5B mutations were located in the SH2 domain, which mediates dimerization and activation by trans-phosphotyrosine binding. The same three STAT5B mutations were also found in the diagnostic sample and most likely represent founding events in leukemogenesis. The N642H and T648S mutations occurred on the same allele with tumor mutation frequencies of approximately 40% while the I704L mutation occurred on a different allele with a similar tumor mutation frequency. To investigate the prevalence of STAT5B mutations in T-ALL we sequenced 17 BM samples from T-ALL patients. In this cohort we could not detect any other patients carrying mutations in the STAT5B SH2 domain. Western blot analysis made with mutant constructs showed that the N642H and I704L mutations induced constitutive phosphorylation of STAT5B. Compared to wild type STAT5B the N642H and I704L mutants induced 47- and 6-fold increases in transcriptional activity, respectively, while T648S mutation had no effect in the assays. The construct with both the N642H and T648S mutations showed the highest amount of constitutive phosphorylation and induced a 56-fold increase in transcriptional activity compared to wild type STAT5B. Using ex vivo drug testing the STAT5B mutated blasts were resistant (EC50≥1 uM) to inhibitors of PI3K (e.g. idelalisib, XL147), dual inhibitors of PI3K/mTOR (PF-04691502, dactolisib) and mTOR inhibitors (temsirolimus, everolimus). Furthermore the blasts showed no response to AKT1 inhibitors (MK-2220) or JAK inhibitors (ruxolitinb, tofacitinib). In contrast, the cells were most sensitive to the BCL-2/BCL-XL inhibitor navitoclax (EC50 83 nM). Summary STAT5B mutations are uncommon in T-ALL but their occurrence underlines the significance of the IL7R-JAK-STAT5 pathway in the pathogenesis of T-ALL. While STAT5B mutant blasts were not sensitive to inhibitors targeting JAK kinases, the cells were unusually sensitive to inhibitors of target molecules of STAT5B, including anti-apoptotic BCL-2 proteins. These results suggest that BCL-2/BCL-XL inhibitors such as navitoclax are novel candidate therapies for T-ALL patients. Disclosures: Mustjoki: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Porkka:BMS: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau.

Beschreibung: Introduction T cell acute lymphoblastic leukaemia (T-ALL) is an aggressive haematological malignancy affecting 10-15% of pediatric ALL patients. Current cure rates of pediatric patients is 80% but increasing understanding of the molecular mechanisms of T-ALL provides possibilities for more effective and targeted therapies. During the last decade targeted therapies with tyrosine kinase inhibitors (TKI) have proven to be effective in BCR-ABL1 fusion positive leukemias. Several studies have suggested TKI dasatinib to be effective also in the treatment of NUP214-ABL1 positive T-ALLs, which comprise approximately 4-10% of the T-ALL cases. Materials and Methods In silico drug screening was performed by comparing gene expression profiles of 4769 leukemic samples to a library of 13384 compounds and their known targets from the Drug signature database (DsigDB). These compounds included FDA approved therapeutic molecules and molecules under studies. Findings were validated in an ex vivo drug screen, consisting of 20 T-ALL bone marrow samples and 9 healthy bone marrow controls. Samples were treated for 72 hours with five different concentrations of dasatinib in 10-fold dilutions (0.1-1000nM), cell viability was measured and the data normalized to negative (DMSO) and positive (benzethonium chloride) controls. The effect of dasatinib was further explored in vitro by treating one NUP214-ABL1 fusion positive and six fusion negative T-ALL cell lines with dasatinib (1-1000nM). Gene expression levels of the known dasatinib targets in these cell lines were measured by Global Run On sequencing (GRO-seq) assay and qRT-PCR. Results In order to find novel targeted therapies for T-ALL, we performed an in silico drug target screen. A dasatinib-targetable gene LCK was strongly expressed in a number of T-ALL cases whereas normal T-lymphoid cells had lower expression. Chemical screen data of the target specificity of dasatinib showed high inhibition of LCK with percent of control (POC) value of 1, meaning that 0.1µM concentration of dasatinib decreases the kinase activity of LCK to 1% in comparison to control. In vitro dasatinib decreased cell viability in fusion negative Jurkat and MOLT-16 cells, and also in fusion positive cell line Peer. GRO-seq and qRT-PCR confirmed the expression of LCK and several other known dasatinib targets, including other SRC family kinases, in Jurkat and MOLT-16 cell lines. However, some LCK-expressing T-ALL cell lines were less sensitive to dasatinib. In further validation, ex vivo drug testing of patient samples revealed a marked response in 6/20 patient samples with IC50 values ranging between 1.3 - 8.2nM, while in healthy bone marrow controls IC50 values were 〉1000nM. Conclusion Our in silico drug screen identified dasatinib as a potential targeted therapy for a subgroup of T-ALL cases, and this finding was further supported by both ex vivo and in vitro studies. The exact mechanism remains to be elucidated but a number of SRC family kinases, which could potentially be targeted by dasatinib, showed expression in T-ALL samples. Disclosures Heckman: Celgene: Research Funding; Pfizer: Research Funding.

Beschreibung: Introduction Many drug discovery efforts and pharmacogenomic studies are based on testing established cancer cell lines for their sensitivity to a given drug or a panel of drugs. This approach has been criticized due to high selectivity and fast proliferation rate of cancer cell lines. To explore new therapeutic avenues for acute myeloid leukemia (AML) and to compare experimental model systems, we applied high-throughput Drug Sensitivity and Resistance Testing (DSRT) platform with 305 approved and investigational drugs for 28 established AML cell lines and compared their drug responses with our earlier study of 28 ex vivo AML patient samples (Pemovska et al., 2013). We then correlated drug sensitivities with genomic and molecular profiles of the samples. Methods DSRT was carried out with 305 clinical, emerging and experimental drugs and small molecule chemical inhibitors. The drugs were tested at five different concentrations over a 10,000-fold concentration range. Cell viability was measured after 72 hours using Cell Titre Glow assay. IC50 values were calculated with Dotmatics software and drug sensitivity scores (DSS, a modified area under the curve metric) were derived for each drug (Yadav et al., 2014). Nimblegen's SeqCap EZ Designs Comprehensive Cancer Design kit was used to identify mutations from 578 oncogenes in cell lines. Results The 28 established AML cell lines were in general more sensitive to the drugs as compared to the 28 ex vivo patient samples, with some important exceptions. Sensitivity towards many targeted drugs was observed in both AML cell lines and in patient samples. These included inhibitors of MEK (e.g. trametinib in 56% of cell lines and 36% of ex vivo samples), mTOR (e.g. temsirolimus in 42% and 32%) and FLT3 (quizartinib in 28% and 18%). Overall, drug responses between cell lines and ex vivo patient cells in AML showed an overall correlation coefficient of r=0.81. BCL2 inhibitors (venetoclax and navitoclax) showed more sensitivity in ex vivo patient cells than in AML cancer cell lines, whereas responses to anti-mitotic agents (docetaxel, camptothecin, vincristine) showed stronger responses in cell lines (Figure). Only 7% of AML cell lines exhibited responses to a broad-spectrum tyrosine kinase inhibitor dasatinib, in contrast to 36% patient samples. AML cell lines that carried FLT3 mutations showed high sensitivity to FLT3 inhibitors. Similarly, cell lines harbouring mutations in RAS or RAF were strongly sensitive to MEK inhibitors. MEK and FLT3 inhibitor responses were mutually exclusive, indicating alternative pathway dependencies in cell lines. However, these pharmacogenomics correlations were not as clearly seen in the clinical samples. Summary These data revealed a few important differences as well as many similarities between established AML cell lines and primary AML patient samples in terms of their response to a panel of cancer drugs. The hope is that patient-derived primary cells in ex vivo testing predict clinical response better as compared to the established cancer cell lines, which indeed seem to overestimate the likelihood of responses to many drugs. On the other hand, cancer cell line studies may also underestimate the potential of dasatinib and BCL2 inhibitors as emerging AML therapeutics. References 1. Pemovska T, Kontro M, Yadav B, Edgren H, Eldfors S, Szwajda A, et al. Individualized systems medicine strategy to tailor treatments for patients with chemorefractory acute myeloid leukemia. Cancer Discovery. 2013 Dec;3(12):1416-29 2. Yadav B, Pemovska T, Szwajda A, Kulesskiy E, Kontro M, Karjalainen R, et al. Quantitative scoring of differential drug sensitivity for individually optimized anticancer therapies. Scientific reports. 2014;4:5193. Figure: Correlation of average drug responses (n=305) between 28 AML cell lines and 28 AML ex vivo patient samples Figure:. Correlation of average drug responses (n=305) between 28 AML cell lines and 28 AML ex vivo patient samples Disclosures Heckman: Celgene: Research Funding. Porkka:BMS: Honoraria; BMS: Research Funding; Novartis: Honoraria; Novartis: Research Funding; Pfizer: Research Funding. Kallioniemi:Medisapiens: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Beschreibung: Background The t(5;11)(q35;p15.5) translocation resulting in fusion of the nucleoporin NUP98 and methyltransferase NSD1 (NUP98-NSD1) genes is a recurrent aberration observed in pediatric and adult AML. The NUP98-NSD1 fusion often co-occurs with the FLT3-ITD mutation and characterizes a group of cytogenetically normal AML patients with very poor prognosis. Despite advances in the understanding of the biology of NUP98-NSD1-positive AML, its therapeutic success rate has remained low. We aimed to identify novel candidate drugs for NUP98-NSD1-positive AML by testing primary patient cells and in vitro cell models with a high-throughput drug sensitivity platform. Methods Leukemic blasts were Ficoll separated from bone marrow (BM) aspirates of an AML patient positive for t(5;11)(q35;p15.5) and FLT3-ITD. RNA extracted from primary cells was used for RNA sequencing and gene expression analysis. NUP98-NSD1 cDNA was amplified from primary cell RNA and expressed from a lentiviral vector (LeGO-iCer2) also encoding the cerulean fluorescent marker. The NUP98-NSD1/LeGo-iCer2 and empty LeGo-iCer2 viruses were used to establish stably expressing Ba/F3 cell lines. Primary murine (BALB/c) BM cells were transduced with NUP98-NSD1 and FLT3-ITD retroviruses alone or in combination (NNF) in vitro (“preleukemic”) or passaged in vivo (“leukemic”) as previously described (Thanasopoulou et al, 2014). For screening, 309 small molecule inhibitors including FDA/EMA-approved and investigational oncology drugs were plated on 384-well plates in a 10,000-fold concentration range. Cells were dispensed on the pre-drugged plates and incubated at 37°C for 72h, and then cell viability measured using the CellTiter-Glo® luminescent assay. Drug response curves were generated and a drug sensitivity score determined (Yadav et al, 2014). Select drug sensitivity was calculated for each drug by comparing results between primary leukemic and healthy donor BM cells or between the cell constructs and empty vector transduced controls cells. Results Primary patient cells and murine BM cells expressing FLT3-ITD alone or in combination with NUP98-NSD1 were selectively sensitive to specific FLT3 inhibitors (e.g. quizartinib, sorafenib and lestaurtinib), and broad-spectrum receptor tyrosine kinase inhibitors targeting FLT3-ITD (e.g. cabozantinib, crenolanib, foretinib, midostaurin, MGCD-265 and ponatinib). Furthermore, these cells were highly sensitive to checkpoint kinase 1/2- inhibitor AZD7762. The primary murine cells expressing both NUP98-NSD1 and FLT3-ITD showed higher sensitivity to all of the above-mentioned drugs compared to cells expressing either of the events alone indicating functional synergy. A very distinct drug response pattern was observed in the leukemic NNF cells cultured in vivo compared to the same cells cultured in vitro suggesting that microenvironment may also affect the observed drug responses. Interestingly, the preleukemic murine cells expressing NUP98-NSD1 with or without FLT3-ITD as well as the primary patient cells showed extreme vulnerability to BCL2/BCL-xL inhibitor navitoclax. Furthermore, primary murine cells expressing NUP98-NSD1 alone showed high select sensitivity to JAK-inhibitors ruxolitinib, BMS-911543, AZD1480 and tofacitinib indicating the fusion may stimulate JAK/STAT-signaling. Similar sensitivity was also observed in the Ba/F3-cells expressing NUP98-NSD1. In support of these findings, gene expression analyses showed high expression of anti-apoptotic factors BCL2, BCL-xL and MCL1 in the patient cells. MCL1 is regulated by STAT3 while BCL-xL is regulated by STAT5, which were also highly expressed. Conclusions In summary, we have observed an enhanced response to specific and non-specific FLT3 inhibitors in cells expressing NUP98-NSD1 and FLT3-ITD together compared to cells expressing either of the two alone. This coincides with previous findings that functional co-operation between NUP98-NSD1 and FLT3-ITD is important in AML (Thanasopoulou et al, 2014). We have seen high in-vitro-in-vivo correlation between primary patient cells and murine cells expressing NUP98-NSD1 and FLT3-ITD. Moreover, we have identified potential candidate compounds targeting oncogenic signaling activated by these two events. These data form a basis for clinical evaluation of candidate compounds for NUP98-NSD1-positive AML. Disclosures Porkka: Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Heckman:Celgene: Research Funding.

Beschreibung: Co-senior authors Andrew Brunner and Andrew H. Wei contributed equally to this work Background: MBG453 is a high-affinity humanized anti-TIM-3 (T-cell immunoglobulin domain and mucin domain-3) IgG4 antibody in development for the treatment of MDS, AML, and other malignancies. TIM-3 is an immune checkpoint with a complex regulatory role in both adaptive and innate immune responses and is also preferentially expressed on leukemic stem and progenitor cells, making it a potential target in MDS and AML. MBG453 has been shown to enhance immune cell-mediated killing of AML cells in vitro. Hypomethylating agents (HMAs) have been shown to increase immune checkpoint expression in MDS and AML, providing rationale to study the combination of HMAs with MBG453. Methods: Patients with Revised International Prognostic Scoring System (IPSS-R) high or very high-risk (HR) MDS and newly diagnosed, or relapsed/refractory (R/R), AML following ≥ 1 prior therapy who were not candidates for standard chemotherapy and who were HMA naive were enrolled in this multi-center, open label phase Ib dose-escalation study (NCT03066648). Escalating doses of MBG453 were administered i.v. every 2 weeks (Q2W; days 8, 22) or every four weeks (Q4W; day 8) in combination with decitabine (20 mg/m2; i.v. days 1-5). The primary objectives were to characterize the safety and tolerability of MBG453 in combination with decitabine and to identify recommended doses for future studies. Secondary objectives included assessing preliminary efficacy and pharmacokinetics of the combination. Dose escalation followed a Bayesian logistic regression model based on dose-limiting toxicities (DLTs). Adverse events (AEs) were graded using NCI-CTCAE v4.03. The International Working Group criteria for MDS (Cheson et al, 2006) or AML (Cheson et al, 2003) were used to assess efficacy. Results: As of March 25, 2019, 17 HR-MDS, 4 chronic myelomonocytic leukemia (CMML), and 38 AML patients have received decitabine and MBG453 at 240 mg Q2W (n=22), 400 mg Q2W (n=21), or 800 mg Q4W (n=16). MTD has not been reached. Median age was 70 years (range 23-87 years). 24 patients are ongoing (duration of exposure 1.1 to 18.6 months) with 35 patients discontinued (disease progression [n=19, 32%], AE [n=1, 2%], patient/physician decision [n=13, 22%], death [n=2, 3%]). There was one DLT consisting of a grade 3 ALT elevation that was corticosteroid responsive. The most common treatment emergent grade 3/4 AEs were febrile neutropenia (39%), neutropenia (34%), thrombocytopenia (31%), and anemia (29%). A total of 8 patients (14%) developed ≥ grade 2 suspected immune related AEs (irAEs) considered to be MBG453 related; 4 of whom (7%) presented with grade 3/4 events: ALT elevation (n=2), arthritis (n=1), and GGT increase (n=1). No study treatment-related deaths were observed. 16 HR-MDS and 31 AML patients have had post-baseline disease response assessments. Median duration of decitabine and MBG453 is 3.9 months (range 0.7-18.6 months). Evidence of activity with MBG453 in combination with decitabine has been seen at doses ranging from 240 mg Q2W to 800 mg Q4W. 8 of 16 (50%) HR-MDS patients achieved mCR or CR. None of the responding HR-MDS patients has had disease recurrence with exposure durations currently ranging from 3.4 to 18.6 months; two patients in mCR underwent allogeneic stem cell transplant. 4 of 14 (29%) newly diagnosed AML patients have achieved a response of PR or better (2 PR, 2 CR), with 3 additional patients exhibiting ≥ 50% bone marrow blast reduction, and 10 of 14 (71%) continuing on study. 5 of 17 (29%) R/R AML patients have achieved a response of CRi, with 5 additional patients exhibiting ≥ 50% bone marrow blast reduction. Exposure durations for all AML responders currently range from 2.1 to 17.9 months. Median onset of response among all patients was 2.0 months. TIM-3 expression was detected on leukemic cells, with modulation of TIM-3 expression following treatment with decitabine. Conclusions: In this ongoing study in patients with HR-MDS and AML, the combination of MBG453 and decitabine was safe and well tolerated, and exhibited evidence of anti-leukemic activity with encouraging preliminary response rates occurring at a median of 2 cycles, with durability in both HR-MDS and AML. These findings validate TIM-3 as a promising therapeutic target in MDS and AML and support further clinical development of MBG453 in combination with HMAs in patients with MDS and AML. Disclosures Borate: AbbVie: Consultancy; Daiichi Sankyo: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Takeda: Consultancy. Esteve:Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Daiichi Sankyo: Consultancy; Celgene: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy; Roche: Consultancy; Astellas: Consultancy, Speakers Bureau; Pfizer: Consultancy. Porkka:Daiichi Sankyo: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Knapper:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; Tolero: Consultancy; Daiichi Sankyo: Honoraria; Pfizer: Consultancy. Vey:Janssen: Honoraria; Novartis: Consultancy, Honoraria. Scholl:Novartis: Other: Project funding; Pfizer: Other: Advisory boards; Gilead: Other: Project funding; AbbVie: Other: Advisory boards; Daiichi Sankyo: Other: Advisory boards. Garcia-Manero:Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Wermke:Novartis: Honoraria, Research Funding. Janssen:Amsterdam University Medical Center, location VUmc, Amsterdam, The Netherlands: Employment; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Other: Founder of the HematologyApp which is supported by BMS, among others, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Founder of the HematologyApp which is supported by Pfizer, among others; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Founder of the HematologyApp which is supported by Incyte, among others; AbbVie: Membership on an entity's Board of Directors or advisory committees; Janssen: Other: Founder of the HematologyApp which is supported by Janssen, among others; MSD: Other: Founder of the HematologyApp which is supported by MSD, among others; Daiichi-Sankyo: Other: Founder of the HematologyApp which is supported by Daiichi-Sankyo, among others; Roche: Other: Founder of the HematologyApp which is supported by Roche, among others; Takeda: Other: Founder of the HematologyApp which is supported by Takeda, among others. Traer:AbbVie: Consultancy; Notable Labs: Equity Ownership; Agios: Consultancy; Astellas: Consultancy; Daiichi Sankyo: Consultancy. Chua:Alfred Hospital, Melbourne, Australia: Employment. Narayan:Takeda: Other: Employment (spouse); Merck: Other: Equity ownership (spouse); Genentech: Other: Equity ownership (spouse). Tovar:Hospital Clinic Barcelona: Employment. Kontro:Amgen: Consultancy; Astellas: Consultancy; AbbVie: Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees. Ottmann:Roche: Honoraria; Pfizer: Honoraria; Fusion Pharma: Honoraria; Takeda: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Sun:Novartis Institutes for BioMedical Research: Employment; Novartis: Other: Novartis stock owner (stock share as long-term employee incentive). Longmire:Novartis Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Szpakowski:Novartis Institutes for Biomedical Research: Employment, Other: Novartis Stock. Liao:Novartis: Employment. Patel:Novartis Pharmaceuticals: Employment. Rinne:Novartis: Employment; N-Of-One, Inc: Consultancy. Brunner:Astra Zeneca: Research Funding; Forty Seven Inc: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees. Wei:Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Honoraria, Research Funding; Janssen: Honoraria; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Macrogenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: AHW is a former employee of the Walter and Eliza Hall Institute and receives a fraction of its royalty stream related to venetoclax, Research Funding, Speakers Bureau; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: MBG453 is an investigational anti-TIM-3 antibody that is being evaluated in hematological malignancies and solid tumors