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Oncogene addiction describes the dependence of some cancers on one or a few genes for their survival. Inhibition of the corresponding oncoproteins can lead to dramatic responses. However, in some cases, such as chronic myeloid leukemia (CML), a disease characterized by the presence of the abnormal fusion tyrosine kinase BCR-ABL, cancer stem cells may never acquire addiction to the oncogene that drives disease development. The suggested mechanism(s) for treatment failure include a quiescent stem cell population capable of reinstating disease, high levels of oncoprotein expression, or acquired mutations in the oncogene. In this review, we discuss the evidence for oncogene addiction in several solid tumors and their potential escape mechanism(s) with a particular focus on CML stem cells. Stem Cells 2014;32:1373–1379

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 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 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.

BCR-ABL1 inhibitors have revolutionized treatment of CML patients. However several drawbacks remain, including therapy resistance of T315I-mutated CML and incapability of current drugs to eliminate quiescent CML stem cells warranting development of novel therapies. In addition, drugs with the potential to enhance efficacy of BCR-ABL1 targeting agents could improve treatment of CML patients. Members of the Tyro3, Axl, Mer receptor (TAMR) tyrosine kinase family are abundantly expressed in physiological and malignant hematopoiesis and their ligand Gas6 can support hematopoietic (progenitor) cells. Evidence in the literature indicates that Axl is upregulated upon treatment with imatinib (IM). In this study, 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 (former designation R428) in primary CML (stem cell) samples, cell lines and preclinical models. In a first step we quantified Axl-expressing cells by flow cytometry in chronic phase (CP) CML bone marrow at primary diagnosis and healthy bone marrow donors. Here, we found higher numbers of Axl-positive cells in CML bone marrow compared to controls (11.42±0.42% (n=5) vs. 0.65±0.10% (n=6), respectively; p=0.0015). In addition, we determined Gas6 plasma levels by ELISA in healthy controls and CML patients in CP and blast crisis (BC). These analyses revealed that Gas6 plasma levels were upregulated in a stage specific manner (plasma levels of Gas6: healthy controls 1290±684 pg/ml (n=14), CP 3465±405 pg/ml (n=50), BC 10940±3868 pg/ml (n=7); p=0.0001). Thus, the Gas6-Axl axis represents a potential therapeutic target in CML patients. Based on this finding we analyzed efficacy of BGB324 in Axl-expressing BV173, KCL22, K562, BaF3_BCR-ABL1-wt and BaF3_BCR-ABL1-T315I cell lines in vitro. We found inhibition of proliferation in all analyzed cell lines with IC50 values ranging from 500-3000 nM. Combination experiments with the IC50 dose of BGB324 and IM revealed additive effects of both treatments in all cell lines. Dose finding experiments with BGB324 in sorted CD34+ primary CML cells grown in the presence of physiological growth factors yielded a mean IC50 of 1.1 ± 0.3 mM (n=3), which was similar to nilotinib. BGB324 did not accumulate CD34+CFSEmax (undivided) cells any more than nilotinib but enhanced apoptosis of CD34+ cells in combination with nilotinib. Notably, there was a consistent inhibitory effect of 3 mM BGB324 alone or in combination with nilotinib against colony forming cells (CFC) with the more primitive BFU-E and GEMM being most sensitive to inhibition (n=4). Interestingly, the Ph- lymphocytes (confirmed by FISH) sorted simultaneously with Ph+ CD34+ cells from the same CML patient were unaffected in terms of viability when treated with Axl inhibitor; thus the BGB324’s activity is cell context specific. Encouraged by these data we analyzed efficacy of BGB324 in an aggressive preclinical CML model in which bone marrow cells were retrovirally transduced with constructs containing T315I-mutated BCR-ABL1. Transduced cells were subsequently i.v. transplanted into sublethally irradiated recipient mice, who rapidly developed blast crisis CML. Mice were treated with 25 mg BGB324 or vehicle BID by oral gavage starting from day 3 after transplantation when homing of transduced cells to the bone marrow is completed. In this model we found a significant prolongation of survival upon treatment with BGB324 (Figure 1). Analyses of the leukemia phenotype by differential blood counts and flow cytometry revealed that BGB324 reduced leukemia cell burden (WBC 138±20x103/µl (n=14) vs. 60x103±21 k/µl WBC in the BGB324 treated group (n=13); p=0.0078). Taken together, these data suggest that the Gas6-Axl axis represents a therapeutic target and that BGB324 is a potent molecule effective against T315I-mutated and wt CML alone and in combination with TKI. Furthermore, BGB324 induces apoptosis of quiescent Ph+ CML stem/progenitor cells. Thus BGB324 might open up novel therapeutic avenues in CML patients. Disclosures: Loges: BerGenBio: research support Other.