Mice expressing a transgene encoding the transcription factor NF-E2 in hematopoietic cells exhibit features of myeloproliferative neoplasms, including thrombocytosis, Epo-independent colony formation, stem and progenitor cell overabundance, leukocytosis, and progression to acute myeloid leukemia.

BCR-ABL1 inhibitors have revolutionized the treatment of CML patients. However several drawbacks remain, including clinical resistance of T315I-mutated CML. Further, the clinical success of ponatinib, a selective inhibitor of T315I-mutated BCR-ABL1 is hampered by vascular side effects. Therefore, novel treatment strategies are warranted especially in T315I-mutated CML. We investigated the relevance of the Gas6-Axl axis in CML patients and the therapeutic potential of the clinically applicable small molecule Axl inhibitor BGB324 in primary CML (stem cell) samples, cell lines and preclinical models. We previously found that Gas6 and Axl represent potential novel targets in this disease and that BGB324 inhibits CML growth in vitro and in vivo (Erdmann et al., ASH meeting 2013, New Orleans, #1469). We next wished to confirm Axl as specific therapeutic target and therefore down regulated its expression in KCL-22 and K562 cells by means of shRNA. In these experiments blockade of Axl inhibited CML cell proliferation in comparison to control-transduced cells, thereby confirming that Axl promotes CML growth. Subsequently, we added BGB324 to shAxl- and shcontrol-transduced cells. Surprisingly BGB324 inhibited cell growth significantly more in shAxl-transduced cells in comparison to control-treated shAxl-transduced cells (p<0.05). These experiments indicated that BGB324 was inhibiting an additional target which supported CML cell proliferation, besides Axl. In order to identify this target we carried out a Kinome Scan revealing that BGB324 binds to native and mutated ABL1. Interestingly, the affinity of BGB324 for ABL1 carrying different mutations including T315I was 5 to 50 fold higher compared to unmutated ABL1 (Table 1). Next, we incubated BaF3 cells stably transfected with BCR-ABL1p210, BCR-ABL1T315I, BCR-ABL1M351T and BCR-ABL1E255K with various concentrations of BGB324 in order to determine its IC50 in the different cell lines. These experiments showed in concordance with the Kinome Scan that BGB324 was more potently inhibiting growth of mutated BCR-ABL1 compared to BCR-ABL1p210 (IC50 BCR-ABL1p210 1266 ± 126 nM; BCR-ABL1T315I 726 ± 194 nM; BCR-ABL1M351T 847 ± 10 nM and BCR-ABL1E255K 794 ± 39 nM; n=2-3; p<0.05 compared to BCR-ABL1p210). Notably, further experiments revealed that BGB324 inhibited KCL-22 cells and K526 cells to a similar extent compared to the combination of imatinib (IM) and shAxl. Thus, BGB324 is a dual inhibitor of BCR-ABL1 and Axl. As the inhibition of BCR-ABL1T315I is of special clinical interest we wished to confirm this finding further in vivo. Therefore we inoculated BCR-ABL1p210 and BCR-ABL1T315I cells subcutaneously into NSG mice. After the tumors reached a size of 80-100 mm3 mice were randomized to receive either placebo control or 50 mg/kg BGB324 delivered twice daily by oral gavage. This experiment showed potent inhibition of tumor growth after 12 days with higher activity of BGB324 in mice bearing BCR-ABL1T315I tumors (placebo: 1751 ± 606 mm3, BGB324: 614 ± 224 mm3; p=0.001) compared to mice bearing BCR-ABL1p210 tumors (placebo: 1432 ± 403 mm3; BGB324: 632 ± 229 mm3; p=0.05) (Figure 1). Subsequently, tissue harvested at end-stage was subjected to immunohistochemical staining for the proliferation marker phospho-histone H3 and Western Blot analyses of cleaved caspase 3 in order to determine whether reduced proliferation and/or increased apoptosis was responsible for reduced growth of BCR-ABL1T315I tumors upon treatment with BGB324. These analyses revealed that proliferation as determined by histomorphometric analysis of phosho-histone H3 was reduced while cleaved caspase 3 levels were unchanged. These data were further corroborated by the finding that treatment with BGB324 reduced the level of phosphorylated MapK as determined by immunoblotting and densitometry. Thus, BGB324 inhibits proliferation of BCR-ABL1T315I cells in vivo. Altogether, our findings show that BGB324 represents a dual inhibitor of Axl and ABL kinase with therapeutic potential in CML, in particular in BCR-ABL1T315I disease. As BGB324 was shown to be well tolerated in healthy volunteers (Wnuk-Lipinska et al., AACR meeting 2013 San Diego #1747), our findings pave the way for clinical investigation of BGB324 in (T315I-mutated) CML. Table 1 Gene KinomeScan Kd (nM) KinaseProfiler IC50 (nM) Axl 0.4 3 ABL1 51.88 51 ABL1(E255K) 1.15 n/a ABL1(T315I) 10.13 4 ABL1(Y253F) 18.19 26 Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Loges: BerGenBio: Research Funding, travel support, advisory boards Other.

961 The transcription factor nuclear factor erythroid-2 (NF-E2) is expressed in hematopoietic stem cells as well as in myeloid, erythroid and megakaryocytic precursors. NF-E2 deficient mice display marked anemia at birth and die perinatally due to thrombopenia, demonstrating an essential role for NF-E2 in both in erythropoiesis and platelet formation. We have previously shown that NF-E2 is overexpressed in the vast majority of patients with Myeloproliferative Neoplasms (MPNs). However, the effect of augmented transcription factor activity has not been studied in vivo. We therefore engineered two independent transgenic mouse lines expressing human NF-E2 under the control of the vav-Promoter, which has previously been shown to direct transgene expression in hematopoietic stem cells as well as in precursor cells of all lineages. The two founder lines differed in the degree of NF-E2 overexpression displayed. While one line showed moderate overexpression (2 – 5-fold), the other line expressed human NF-E2 between 10 and 100-fold above the murine counterpart. Both lines paralleled observations in PV patients, where a wide range of NF-E2 overexpression was noted (median overexpression, 7-fold; range 2-fold to 40-fold; n = 59). The two founder lines show overlapping but distinct phenotypes. In both strains. moderately overexpressing NF-E2 transgenic mice (2 – 10-fold) invariably develop thrombocytosis with a latency of 14 months. In addition, megakaryocyte colony formation in the bone marrow is drastically increased. In contrast, thrombocytosis is not observed in the markedly overexpressing NF-E2 transgenic mice (above 20-fold). A similar inverse correlation between the degree of NF-E2 overexpression and platelet numbers was observed in MPN patients. In both strains, Epo-independent colony formation, a pathognomonic feature of polycythemia vera, is significantly increased in NF-E2 transgenic animals. Bone marrow histopathology shows findings characteristically seen in MPNs, including the presence of increased megakaryopoiesis with cytologically abnormal forms, often in clusters. Both NF-E2 transgenic strains display significantly increased mortality. Upon autopsy, between 15 and 20% of mice in both strains present with major gastrointestinal bleeding in conjunction with splenic atrophy. Spleen weight is reduced by over 50% (Transgenic mice: 49 +/-15 mg, wild type littermates 103 +/- 30 mg; p < 0.001, n = 8 each). One third of the remaining mice show moderate to marked splenomegaly (2 – 27 fold increase in spleen weight; mean: 434 mg, range: 124 – 2700 mg; p < 0.001 vs. wt littermates, n = 12). Histopathological examination of all spleens revealed mild to moderately expanded red pulp with increased numbers of iron containing histiocytes. This observation indicates increased red cell destruction and may explain the fact that neither hematocrit nor hemoglobin are elevated in NF-E2 transgenic animals. At 18 months of age, one mouse developed acute leukemia, which is currently being phenotyped. In summary, in a murine model moderate NF-E2 overexpression causes a phenotype resembling Essential Thrombocythemia. In addition, our preliminary data indicate that NF-E2 overexpression may predispose to the development of acute leukemia. Disclosures: No relevant conflicts of interest to declare.