ALBERT

Description: Acute myeloid leukemias (AML) are characterized by recurrent genomic alterations, often in transcriptional regulators, which form the basis on which current prognostication and therapeutic intervention is overlaid. Three subtypes of AML carrying specific translocations, namely t(15;17), t(11;17) and t(6;9), are notable for being associated with a smaller number of co-existing driver mutations than e.g. AML with normal karyotype. This strongly suggests that the function of their aberrant gene products, PML/RAR and DEK/CAN, respectively, may subsume the functions of other driver mutations. Thus we hypothesized that these functions, while as yet elusive, not necessarily require sequential acquisition of secondary genomic alterations. We elected to study AML with the t(6;9), defined as a distinct entity by the WHO classification, because of its particular biological and high risk clinical features and unmet clinical needs. Most t(6;9)-AML patients are young, with a median age of 23-40 years, complete remission rates do not exceed 50% and median survival after diagnosis is only about 1 year. We used a novel "subtractive interaction proteomics" (SIP) approach to understand the mechanisms by which the t(6;9)-DEK/CAN nuclear oncogene induces this highly resistant leukemic phenotype. Based on Tandem Affinity Precipitation (TAP) for the enrichment of proteins complexes associated with SILAC-technology followed by LC-MS/MS we developed SIP as a comparison between the interactome of an oncogene and those of its functionally inactive mutants in order to obtain eventually only relevant interaction partners (exclusive binders) in the same genetic background. This is achieved by the subtraction of binders that are common to four functionally inactive mutants classifying them as not relevant. Bioinformatic network analysis of the 9 exclusive binders of DEK/CAN revealed by SIP (RAB1A, RAB6A, S100A7, PCBD1, Clusterin, RPS14 and 19, IDH3A, SerpinB3) using BioGrid, IntAct and String together with Ingenuity© Pathway Analysis (IPA), indicated a functional relationship with ABL1-, AKT/mTOR-, MYC- and SRC family kinases-dependent signaling. Interestingly, we found all these signaling pathways strongly activated in an autonomous manner in four DEK/CAN-positive leukemia models, DEK/CAN expressing U937 cells, t(6;9)-positive FKH-1 cells, primary syngeneic murine DEK/CAN-driven leukemias, and t(6;9)-positive patient samples. Bioinformatic analysis of the phopshoproteomic profile of FKH1 cells upon molecular targeting of single pathways (imatinib for ABL1, PP2 for SFKs, dasatinib for ABL1/SFK and Torin1 or NVP-BEZ-235 for mTOR/AKT) revealed that these signaling pathways were organized in clusters creating a network with nodes that are credible candidates for combinatorial therapeutic interventions. On the other hand inhibition of individual outputs had the potential to activate interconnected pathways in a detrimental manner with consequential clinical impact e.g. the activation of STAT5 by the inhibition of mTOR/AKT in these cells. Treatment of mice injected with primary syngeneic DEK/CAN-induced leukemic cells with dasatinib (10mg/kg) and NVP-BEZ-235 (45mg/kg) alone and in combination for 14 days led to a strong reduction of leukemia burden in all cohorts (each cohort n=7). In fact, as compared to untreated controls (146.6 +/- 36mg), mice treated with NVP-BEZ 235 alone and in combination (61.7 +/-4.7mg and 65.3+/- 4.6mg, respectively) showed a statistically significant reduction of spleen size whereas those treated with dasatinib alone (77.5 8 +/- 5.4mg) did not reach statistical significance. Taken together the here presented results reveal specific interdependencies between a nuclear oncogene and kinase driven cancer signaling pathways providing a foundation for the design of therapeutic strategies to better address the complexity of cancer signaling. In addition, it provides evidence for the need of a more in depth analysis of indirect effects of molecular targeting strategies in a preclinical setting not only in AML but in all cancer types. Disclosures Ottmann: Novartis: Consultancy; Pfizer: Consultancy; Fusion Pharma: Consultancy, Research Funding; Amgen: Consultancy; Celgene: Consultancy, Research Funding; Takeda: Consultancy; Incyte: Consultancy, Research Funding.

Description: Introduction: The t(9;22) (q34;q11) translocation results in the constative active BCR/ABL tyrosine kinase. Der22 involves the Breakpoint Cluster Region (BCR) gene locus with two principal breaks: a. M-bcr, encoding for the p210-BCR/ABL and b. m-bcr, encoding for the p185-BCR/ABL fusion proteins, respectively. BCR/ABL is the oncogenic driver of Chronic Myeloid Leukemia (CML) and 30% of adult Acute Lymphatic Leukemia (ALL). Activated BCR/ABL kinase is responsible for aberrant activation of multiple signaling pathways, such as JAK/STAT, PI3K/AKT and RAS/MAPK which eventually result in leukemic transformation. Successful targeting of BCR/ABL by selective tyrosine kinase inhibitors (TKIs) such as Imatinib, Nilotinib, Dasatinib and Ponatinib are used for the treatment of Philadelphia chromosome-positive (Ph+) leukemias. Most patients with CML in the early stage (CML-CP) treated with TKIs have increased overall survival. However, TKIs have not been as effective in patients with CML blast crisis (CML-BC) or Ph+ ALL. Point mutations in the tyrosine kinase domain (TKD) of BCR/ABL have emerged as the predominant cause of acquired resistance. These mutations are observed in up to 80% of patients with CML-BC and Ph+ ALL and in ~ 50% of Imatinib-resistant patients. In the remaining 20-50% of patients the mechanism of resistance to TKIs remains elusive. The aim of this study was to investigate the mechanism of non-mutational resistance in Ph+ ALL. Methods: As models for non-mutational resistance, we used patient derived long term cultures (PDLTCs) from Ph+ ALL patients with different levels of non-mutational drug resistance and the SupB15RT, a Ph+ ALL cell-line rendered resistant by exposure to increasing doses of Imatinib and cross-resistant against all approved ABL Kinase Inhibitors (AKIs). Cell proliferation was assessed by XTT/MTT and trypan blue dye exclusion. Signaling pathway proteins were assessed by Western Blot analysis. Chromosomal karyotyping was undertaken on single cell genomes using multi-color FISH (M-FISH) technology. Mutation analysis on the ABL kinase domain was done by sequencing the heminested PCR products obtained from SupB15-WT and SupB15RT cell-lines. Results: A non-mutational resistance cell line SupB15RT, was developed by exposing SupB15 cells to an increasing concentration of Imatinib over a 3 month period. SupB15RT were able to grow in 10 µM Imatinib. SupB15RT cells were karyotypically and mutationaly identical to SupB15 WT. All approved AKIs and allosteric inhibitors like GNF-2, ABL001 and Crizotinib were unable to inhibit growth of these cells, except for Dasatinib (IC50 40nM), a multi-target kinase inhibitor. Experiments to determine the mode of resistance revealed high level (3 fold) of activation of AKT/mTOR enabling these cells to grow and proliferate. We targeted the AKT/mTOR pathway using BKM-120 (PI3 Kinase inhibitor), BEZ-235 (PI3 Kinase and mTOR pathway) and Trorin1/Torin2 (mTORC1 and mTORC2) and found that Torin-1 and Torin-2 significantly inhibited proliferation of SupB15RT, in a dose dependent manner, with an IC50 of 11-20 nM. As Dasatinib alone inhibited growth of SupB15RT cells at 40-50nm concentrations, we combined Dasatinib with Torin1 and found that the combination of these two compounds had an additive inhibitory effect on cell growth. Following this we examined clinical samples from patients. We used three different Ph+ PDLTCs: a. HP (BCR/ABL negative), b. PH (BCR/ABL positive and responsive to TKIs) and c. BV (BCR/ABL positive and non-mutational resistant to TKIs). Interestingly, we found that AKT/mTOR pathway was activated in BV cells and its proliferation was inhibited by Torin1 with IC-50 of 50nM. Conclusion: Our experiments revealed an additional pathway involved in the evolution of non-mutational resistance in Ph+ ALL which could assist in developing novel targeted therapy for Ph+ ALL patient(s) with non-mutational resistance. Disclosures Ottmann: Celgene: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria; Fusion Pharma: Honoraria; Pfizer: Honoraria; Roche: Honoraria.

Description: Resistance to therapy including potent and selective targeted agents remains the major clinical challenge in AML. In fact, allogeneic stem cell transplantation remains the best curative treatment option for AML patients with high-risk features. This unmet clinical need may be addressed by novel approaches based on targeting networks of activated cancer signaling pathways (NACSPs) instead of only individual pathways. Signaling pathway activation in AML is not only related to class I mutations such as FLT3-ITD, but also to class II driver mutations. This is best exemplified by AML subtypes harboring non-random chromosomal aberrations, encoding driver mutations able to induce and maintain leukemia. Despite the contribution of class I mutations to a poor prognosis, targeting these lesions does not decisively contribute to the cure of AML in the great majority of the cases. AML with the translocation t(6;9)(p23;q34) encoding the related DEK/NUP214 fusion protein is a high risk group of AML patients characterized by young age and presence of FLT3-ITD in ~ 75% of the cases, which responds only transiently to FLT3-ITD inhibitors. Using this AML subtype as a model for high risk disease, we investigated the NACSPs activated by a class II driver mutation. In these patients FLT3-ITD represents the only established recurrent genetic aberration at diagnosis in addition to the t(6;9). We have shown that the driver mutation DEK/NUP214 transforms very immature hematopoietic stem cells with the contribution of activated STAT5, present also in FLT3-ITD-negative patients. Furthermore it has been reported that AKT/mTOR is activated in DEK/NUP214-positive cells. Here we investigated whether these signaling pathways are components of a leukemogenic NACSP and are therapeutically/clinically significant. We used different inhibitors to target either selectively or to determine a inhibition pattern of a.) PI3K/AKT/mTOR signaling (BKM120, BEZ2315, RAD001, Torin1 and AZD1208) at different levels; b.) receptor tyrosine kinases (RTK- ruxolitinib, sorafenib); and c.) members of the SRC kinase family (dasatinib, ponatinib, PF114, PP2). We employed four different models of t(6;9)-positive AML: U937 cells stably transfected with DEK/NUP214, t(6;9)-positive FKH1 cells, syngeneic DEK/NUP214-driven murine AML cells and primary t(6,9)-positive AML cells. Here we show that i.) STAT5 and AKT/mTOR activation was genetically determined by the t(6;9)-DEK/NUP214; ii.) STAT5 and AKT/mTOR activation were independent of JAK2 and PI3K activation, respectively; iii) selective inhibition of the AKT/mTOR cascades strongly increased STAT5 activation; iv.) both signaling pathways form a NACP, with activated members of the SRC kinase family (SKF - LYN and SRC) as a central node; v.) the NACSP was effectively targeted by inhibitors of the SRC-kinase activity (SKI) such as dasatinib, ponatinib, PF114 and the selective SKF inhibitor PP2, resulting in cell growth arrest and induction of apoptosis in t(6;9)-positive leukemic cells; vi.) SKI not only inhibited SKF/STAT5 but also the AKT/mTOR cascade; vii.) this NACP was independent of the activation of RTKs such as PDGFR, KDR, c-KIT, FLT3 a.o, as part of the target profile of many SKIs used above, as shown by the lack of activity of inhibitors such as ruxolitinib, sorafenib or ibrutinib; viii.) addition of AKT/mTOR inhibitors strongly increased effects of low dose dasatinib or ponatinib in primary t(6;9) leukaemic cells. Our findings implicate the t(6;9)-DEK/NUP214 oncogene as a central inductor of an NACSP including SFK, AKT/mTOR and STAT5 which is independent of the recurrent FLT3-ITD signaling in these patients. This NACSP is able to maintain the leukemia in the presence of effective inhibition of FLT3-ITD signaling by Sorafenib, AC220 and other inhibitors in clinical use. Furthermore, the increase of STAT5 activation upon AKT/mTOR inhibition, suggests that the use of such a treatment would not lead to the eradication of the disease, because of the role of activated STAT5 in the maintenance of leukemic stem cells. On the other hand, SKIs target the entire NACSP. In fact, hitting the central node of the NACSP abolished the activation of both STAT5 and AKT/mTOR. Taken together these data establish SKIs as a valid therapeutic concept not only in t(6;9)-positive AMLs but also in all other AML subtypes characterised by the same NACSP. Disclosures Ottmann: Pfizer: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Fusion Pharma: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Ariad: Consultancy, Honoraria.