ALBERT

Description: 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.

Description: 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

Description: 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.

Description: 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.

Description: Introduction T-PLL is a rare mature post-thymic T-cell neoplasm with an aggressive clinical course and median overall survival of less than one year. Almost 75% of T-PLL cases harbor chromosome 14 translocations involving the T-cell receptor A/D locus resulting in aberrant activation of the proto-oncogenes TCL1A or MTCP1. T-PLL patients are difficult to treat as the leukemic cells are often resistant to most available chemotherapeutic drugs. Due to the rareness and aggressive nature of the disease, large clinical trials are difficult to execute. We therefore aimed to discover novel potential therapeutic targets using a high-throughput ex vivo drug sensitivity and resistance testing (DSRT) platform covering 306 approved and investigational oncology drugs. Methods Primary T-PLL cells were available from two patients. The first patient had a double positive CD4+CD8+CD3+ Vβ.14+ T-cell phenotype (patient 1) and cells underwent DSRT twice during a 5-month time-period (no treatment during that time). The second patient had a CD4+CD3+ phenotype (patient 2) and the cells were assayed once by DSRT. Fresh blood mononuclear cells (MNCs) were separated by Ficoll centrifugation from the patient samples (over 85 % leukemic cells in the MNC fraction) and healthy controls. Cells were seeded in 384-well plates and 306 active substances were tested using a 10,000-fold concentration range resulting in a dose-response curve for each compound. Cell viability was measured after 72 h incubation and differential drug sensitivity scores (DSS), representing leukemia-specific responses, were calculated by comparing patient samples with those obtained from healthy donors. In addition, both exome and RNA sequencing was performed from T-PLL cells (patient 1). Results Both patient samples showed high sensitivity to small molecule BCL2-inhibitors navitoclax (EC50 values 44nM and 10nM) and ABT-199 (EC50 23nM and 20nM) (Fig. 1 and 2). HDAC-inhibitors (quisinostat, belinostat and panobinostat) also showed high sensitivity in both patients in low nM concentrations (EC50 values 1-80nM). As AKT1/mTOR pathway is activated in most T-PLL patients due to the TCL1 oncoprotein, it was interesting to observe that neither of the patient samples showed any response to an AKT1 inhibitor (MK-2206 EC50 values 〉1000 nM) nor to mTOR inhibitors (temsirolimus and everolimus)(Fig. 1). Furthermore, T-PLL cells were resistant to corticosteroids such as prednisolone and methylprednisolone. To further elucidate the molecular mechanism behind the drug responses, exome and RNA sequencing was performed from T-PLL cells (patient 1). No deletion was found in the ATM gene, but instead a homozygous missense mutation K2413Q was detected. This particular mutation is in the region coding for the FAT domain and while it has not been described earlier in T-PLL, it is in a cancer mutation hotspot region of ATM, suggesting that it is inactivating. No mutations directly linked to the BCL2-family genes were observed. In the RNA sequencing analysis, TCL1A was overexpressed when compared to the healthy CD4+ cells as expected. Similarly, AKT1 was overexpressed. The expression of BCL-2 and BCL-XL did not differ from those observed in healthy CD4+ cells while pro-apoptotic BCL-2 family members BID and BAD were elevated compared to the healthy control. Conclusions Primary T-PLL cells showed sensitivity to BCL-2 and HDAC inhibitors in a systematic high-throughput ex vivo drug sensitivity testing across a range of clinical and investigational drugs. The BCL-2 inhibitor sensitivity was not related to increased BCL-2 expression or activating mutations in the BCL-2 family genes, and further studies are needed to clarify the mechanism of action. However, the results suggest that BCL-2 inhibitors could be a novel promising candidate drug for T-PLL-patients and warrant further clinical development in this group of patients. In contrast, inhibitors of AKT and mTOR, kinases known to be activated by TCL1, showed no efficacy ex vivo in this assay. Disclosures: Porkka: BMS: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau. Mustjoki:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau.

Description: Introduction T-PLL is a mature post-thymic T-cell neoplasm with an aggressive clinical course (5-year overall survival 21%). Almost 75% of T-PLL cases harbor chromosome 14 translocations resulting in aberrant activation of the proto-oncogene TCL1A. Furthermore, in the majority of T-PLL cases the ATM gene is mutated or deleted, and recently it was reported that mutations in genes involved in the JAK-STAT pathway were found in 76% of T-PLL cases. Due to the rareness and aggressive nature of the disease, clinical trials are difficult to execute. By using a high-throughput ex vivo drug sensitivity and resistance testing (DSRT) platform covering 306 approved and investigational oncology drugs we systematically investigated the heterogeneity of drug responses in PLL-patients. As the impact of mutations on drug sensitivity is not well understood we aimed to identify relevant associations between the drug responses and genetic lesions in T-PLL patients. Methods Primary cells (MNCs) from seven T-PLL patients were obtained for drug screening. Samples were seeded in 384-well plates and 306 active substances were tested using a 10,000-fold concentration range resulting in a dose-response curve for each compound. Cell viability was measured after 72 h incubation and differential drug sensitivity scores (sDSS), representing leukemia-specific responses, were calculated by comparing patient samples to healthy donors. Hierarchical clustering of the drug responses was performed with Cluster 3.0 and Java Tree View. To assess the performance of the drug screening platform we also exchanged six samples with the German Cancer Research Center in Heidelberg for a comparison of results between two independent drug screening systems. To understand heterogeneous pathway dependencies, drug sensitivities were correlated with somatic genetic variants and recurrent chromosomal aberrations. Genetic characterization was performed by exome sequencing of tumor and matched healthy cells to profile known recurrent genetic variants (ATM, STAT5b, IL2RG, JAK1, JAK3) as well as CNVs (TCL1A translocations, ATM deletions, recurrent chromosomal aberrations). Results Four out of seven patient samples showed high sensitivity to small molecule BCL2 inhibitors navitoclax (IC50: 10-68nM) and ABT-199 (IC50: 14-45nM) and to HDAC inhibitors panobinostat and belinostat (IC50: 2-65nM). Intriguingly, the CDK inhibitor SNS-032 was effective in 6/7 patient samples (IC50: 7-95nM). SNS-032 inhibits Cdk2, Cdk7 and Cdk9, which control transcription of anti-apoptotic proteins including MCL1 and XIAP. As the AKT1/MTOR pathway is activated in many T-PLL patients due to expression of the TCL1A oncoprotein, it was interesting to observe that patient samples did not show any response to AKT inhibitors (MK-2206 and GDC-0068 IC50 values 〉1000 nM) nor to MTOR inhibitors (rapalogs temsirolimus and everolimus). Similarly, T-PLL cells were insensitive to JAK-inhibitors. Clustering of drug responses from T-PLL patients with primary AML and ALL patient samples revealed the drug response profiles to be specific for T-PLL patients (Figure). 6/7 patients clustered together while the only patient (PLL4) in our cohort with confirmed mutations in the JAK-STAT pathway genes STAT5b (P702S) and IL2RG (K315E) exhibited a non-sensitive response pattern when compared to other samples (Figure). Interestingly, exome sequencing did not reveal any JAK mutations in our PLL-cohort (n=5) nor additional STAT5b or IL2RG mutations in other patients except in this unresponsive patient. In the comparison between the platforms the correlation of the censored IC50 values from the 60 overlapping drugs was r=0.75. Similar fits of dose-response curves were seen for most drugs, although there were notable exceptions, which may be due to divergent culture conditions and day of read-out. Conclusions Ex vivo drug testing of primary patient cells has the potential to provide novel personalized drug candidates (such as BCL2, HDAC and CDK inhibitors) for T-PLL. The drug response pattern was T-PLL specific warranting further clinical testing. Drug screening, mutation analysis and RNA sequencing of additional patients is currently ongoing (n=20) to validate whether drug responses can be predicted based on the mutation profile or aberrant gene expression. Figure Clustering of T-PLL, AML and ALL patient samples based on DSRT results. Figure. Clustering of T-PLL, AML and ALL patient samples based on DSRT results. Disclosures Kallioniemi: Medisapiens: Consultancy, Membership on an entity's Board of Directors or advisory committees. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Description: 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.

Description: Abstract 288 Introduction: Recent genomic analyses of acute myeloid leukemia (AML) patients have provided new information on mutations contributing to the disease onset and progression. However, the genomic changes are often complex and highly diverse from one patient to another and often not actionable in clinical care. To rapidly identify novel patient-specific therapies, we developed a high-throughput drug sensitivity and resistance testing (DSRT) platform to experimentally validate therapeutic options for individual patients with relapsed AML. By integrating the results with exome and transcriptome sequencing plus proteomic analysis, we were able to define specific drug-sensitive subgroups of patients and explore predictive biomarkers. Methods: Ex vivo DSRT was implemented for 29 samples from 16 adult AML patients at the time of relapse and chemoresistance and from 5 healthy donors. Fresh mononuclear cells from bone marrow aspirates (〉50% blast count) were screened against a comprehensive collection of cytotoxic chemotherapy agents (n=103) and targeted preclinical and clinical drugs (n=100, later 170). The drugs were tested over a 10,000-fold concentration range resulting in a dose-response curve for each compound and each leukemia sample. A leukemia-specific drug sensitivity score (sDSS) was derived from the area under each dose response curve in relation to the total area, and comparing leukemia samples with normal bone marrow results. The turnaround time for the DSRT assay was 4 days. All samples also underwent deep exome (40–100×) and transcriptome sequencing to identify somatic mutations and fusion transcripts, as well as phosphoproteomic array analysis to uncover active cell signaling pathways. Results: The drug sensitivity profiles of AML patient samples differed markedly from healthy bone marrow controls, with leukemia-specific responses mostly observed for molecularly targeted drugs. Individual AML patient samples clustered into distinct subgroups based on their chemoresponse profiles, thus suggesting that the subgroups were driven by distinct signaling pathways. Similarly, compounds clustered based on the response across the samples revealing functional groups of compounds of both expected and unexpected composition. Furthermore, subsets of patient samples stood out as highly sensitive to different compounds. Specifically, dasatinib, rapalogs, MEK inhibitors, ruxolitinib, sunitinib, sorafenib, ponatinib, foretinib and quizartinib were found to be selectively active in 5 (31%), 5 (31%), 4 (25%), 4 (25%), 3 (19%), 3 (19%), 2 (13%), 2 (13%), and 1 (6%) of the AML patients ex vivo, respectively. DSRT assays of serial samples from the same patient at different stages of leukemia progression revealed patterns of resistance to the clinically applied drugs, in conjunction with evidence of dynamic changes in the clonal genomic architecture. Emergence of vulnerabilities to novel pathway inhibitors was seen at the time of drug resistance, suggesting potential combinatorial or successive cycles of drugs to achieve remissions in an increasingly chemorefractory disease. Genomic and molecular profiling of the same patient samples not only highlighted potential biomarkers reflecting the ex vivo DSRT response patterns, but also made it possible to follow in parallel the drug sensitivities and the clonal progression of the disease in serial samples from the same patients. Summary: The comprehensive analysis of drug responses by DSRT in samples from human chemorefractory AML patients revealed a complex pattern of sensitivities to distinct inhibitors. Thus, these results suggest tremendous heterogeneity in drug response patterns and underline the relevance of individual ex vivo drug testing in selecting optimal therapies for patients (personalized medicine). Together with genomic and molecular profiling, the DSRT analysis resulted in a comprehensive view of the drug response landscape and the underlying molecular changes in relapsed AML. These data can readily be translated into the clinic via biomarker-driven stratified clinical trials. Disclosures: Mustjoki: Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria. Kallioniemi:Roche: Research Funding; Medisapiens: Membership on an entity's Board of Directors or advisory committees. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Description: BACKGROUND: BCL-2 family members play a critical role in the regulation of apoptosis. BCL-2 and BCL-XL promote cell survival by preventing mitochondrial apoptotic pore formation. BH3 mimetic drugs such as venetoclax (ABT-199) promote apoptosis by inhibiting BCL-2 while navitoclax (ABT-263) inhibits both BCL-2 and BCL-XL. In AML, the expression of anti-apoptotic proteins is highly variable. In a recent study venetoclax showed single-agent activity in 6/12 AML cell lines and 20/25 patient samples. (Pan et al. Cancer Disc 2014). The samples with complex cytogenetics were largely resistant. Sensitivity correlated with increased BCL-2 protein levels and negatively correlated with BCL-XL and MCL-1 protein levels. We aimed to expand these data to both newly diagnosed and relapsed AML patients and to isolate biomarkers for patient selection. METHODS: We assessed the ex vivo sensitivity of fresh leukemic cells from 16 diagnosed and 36relapsed/refractory AML patient samples to venetoclax (25 samples) and navitoclax (52 samples). Exome sequencing was performed on 32 samples and gene expression of BCL2 family members (BCL-2, BCL-XL, MCL-1, BIK, BAX, BAK1, BID, BCL2L12, BIM, BCL2A1, PUMA and BAD) was determined on 31 samples by qRT-PCR. Samples from primary cells of healthy individuals (n=10), and CMML (n=7) or CLL (n=2) patients were used as controls. Drug sensitivity was determined over a 10,000-fold concentration range (1-10 000 nM). A leukemia-specific drug sensitivity score (sDSS) derived from area under the dose response curve calculations was used as the efficacy variable by comparing leukemia results with those from normal bone marrow cells (Bhagwan et al., Sci Rep 2014, Pemovska et al., Cancer Disc 2014). RESULTS: Compared to healthy controls, CMML samples were largely non-sensitive, whereas CLL samples were highly sensitive to BCL-2 inhibitors ex vivo. The AML samples exhibited heterogeneous responses. 15/25 (60%) of AML samples were sensitive to venetoclax and 35/52 (67%) to navitoclax. Both diagnostic (12 of 16 samples, 75%) and relapsed/refractory samples (24 of 36 samples, 64%) were sensitive to navitoclax. Similarly, 6/7 (86%) of diagnostic samples and 9/18 (50%) of relapsed/refractory samples were sensitive to venetoclax. We observed responses to venetoclax and navitoclax in each patient to be similar, although navitoclax showed efficacy at lower concentrations: in 25 samples tested with both agents, mean sDSS values were lower in navitoclax-treated samples (paired t-test, p=0.02). All except one patient sample exhibited a difference in resistance between the two drugs showing sensitivity to navitoclax but not to venetoclax. We observed responses across all mutational profiles, including samples with mutations to FLT3 -ITD, NPM1, TP53, NRAS and IDH1 and IDH2, as well as in samples with complex karyotypes. Intriguingly, three of four samples with mutated TP53 exhibited sensitivity to BCL2 inhibition. No single mutation predicted sensitivity or resistance. At the RNA level, no statistical correlation between BCL2 or BCL-XL expression for BCL2 inhibitor response was observed. Instead we observed high levels of beta-2-microglobulin (B2M) mRNA expression in BCL2 inhibitor-resistant samples with a strong negative correlation to navitoclax sensitivity (r=-0.60, P=0.0008). DISCUSSION: We did not observe BCL2 and BCL-XL mRNA expression to be optimal predictors for BCL2 inhibitor response. On the other hand, we observed high expression of B2M mRNA expression in resistant samples suggesting that it could serve as a biomarker for sensitivity to BCL2 inhibitors. The high B2M expression has been previously linked to poor prognosis in solid tumors and in AML (Albitar et al., Leukemia 2007). In cell line models B2M leads to phosphorylation and inactivation of proapototic protein BAD (Nokura et al., J Urol 2007). This may affect the balance between pro- and antiapoptotic proteins and thus offer a escape route from BCL2 inhibition. To conclude, we observed BCL2 inhibition to be effective ex vivo in over half of all AML samples, tested both in primary and relapsed/refractory state as well as across different subgroups defined by AML driver mutations. Of the potential biomarkers that were assessed, B2M was the best mRNA-level indicator for anti-BCL-2 drug efficacy. Disclosures Off Label Use: BCL2 inhibitors are not approved for the treatment of AML. Heckman:Celgene: Honoraria, Research Funding; Pfizer: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria.