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Resistance remains the major clinical challenge for the therapy of Philadelphia chromosome–positive (Ph+) leukemia. With the exception of ponatinib, all approved tyrosine kinase inhibitors (TKIs) are unable to inhibit the common “gatekeeper” mutation T315I. Here we investigated the therapeutic potential of crizotinib, a TKI approved for targeting ALK and ROS1 in non-small cell lung cancer patients, which inhibited also the ABL1 kinase in cell-free systems, for the treatment of advanced and therapy-resistant Ph+ leukemia. By inhibiting the BCR-ABL1 kinase, crizotinib efficiently suppressed growth of Ph+ cells without affecting growth of Ph− cells. It was also active in Ph+ patient-derived long-term cultures (PD-LTCs) independently of the responsiveness/resistance to other TKIs. The efficacy of crizotinib was confirmed in vivo in syngeneic mouse models of BCR-ABL1- or BCR-ABL1T315I-driven chronic myeloid leukemia–like disease and in BCR-ABL1-driven acute lymphoblastic leukemia (ALL). Although crizotinib binds to the ATP-binding site, it also allosterically affected the myristol binding pocket, the binding site of GNF2 and asciminib (former ABL001). Therefore, crizotinib has a seemingly unique double mechanism of action, on the ATP-binding site and on the myristoylation binding pocket. These findings strongly suggest the clinical evaluation of crizotinib for the treatment of advanced and therapy-resistant Ph+ leukemia.

Graphical Abstract 2 IDH3A CLU S100A7 PCBD-1 RAB1A RAB6A SerpinB3 M u t a n t 1 DEK/CAN RpS14 RpS19 M u t a n t 2 M u t a n t 3 M u ta n t 4 Subtractive proteomics DEK/CAN SFK MYC mTOR AKT ABL RPS14 RPS19 PCBD1 SerpB3 CLU RAB6A RAB1A S100A7 RAB5A RIN1 IDH3A STAT5 AKT mTOR SRC/SFK ABL c-MYC proliferation self renewal differentiation block apoptosis block therapy resistance DEK/CAN SummaryAcute 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.In AML transformation can often be attributed to single chromosomal aberrations encoding oncogenes, such as t(15;17)-PML/RARα or t(6;9)-DEK/CAN but it is unclear how these aberrant transcription factors drive leukemic signaling and influence cellular responses to targeted therapies. Here we show that by using a novel "subtractive interaction proteomics" approach, the high risk AML-inducing oncogene t(6;9)-DEK/CAN directly activates signaling pathways that are driven by the ABL1, AKT/mTOR, and SRC family kinases. The interplay of these signaling pathways creates a network with nodes that are credible candidates for combinatorial therapeutic interventions. These results reveal specific interdependencies between nuclear oncogenes and cancer signaling pathways thus providing a foundation for the design of therapeutic strategies to better address the complexity of cancer signaling.

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.

The WHO classifies t(6;9)-positive acute myeloid leukemia (AML) as a subgroup of high-risk AML because of its clinical and biological peculiarities, such as young age and therapy resistance. t(6;9) encodes the DEK/NUP214 fusion oncoprotein that targets only a small subpopulation of bone marrow progenitors for leukemic transformation. This distinguishes DEK/NUP214 from other fusion oncoproteins, such as PML/RARα, RUNX1/ETO, or MLL/AF9, which have a broad target population they block differentiation and increase stem cell capacity. A common theme among most leukemogenic fusion proteins is their aberrant localization compared to their wild-type counterparts. Although the actual consequences are widely unknown, it seems to contribute to leukemogenesis most likely by a sequester of interaction partners. Thus, we applied a global approach to studying the consequences of the aberrant localization of t(6;9)-DEK/NUP214 for its interactome. This study aimed to disclose the role of localization of DEK/NUP214 and the related sequester of proteins interacting with DEK/NUP214 for the determination of the biology of t(6;9)-AML. Here we show the complexity of the biological consequences of the expression of DEK/NUP214 by an in-depth bioinformatic analysis of the interactome of DEK/NUP214 and its biologically dead mutants. DEK/NUP214’s interactome points to an essential role for aberrant RNA-regulation and aberrant regulation of apoptosis and leukocyte activation as a significant determinant of the phenotype of t(6;9)-AML. Taken together, we provide evidence that the interactome contributes to the aberrant biology of an oncoprotein, providing opportunities for developing novel targeted therapy approaches.