ALBERT

Description: Abstract 5042 Background In acute myeloid leukemia (AML) a recurrent chromosome abnormality t(12;15)(p13;q25) fuses ETV6 with NTRK3. This rearrangement uniquely occurs in both solid tumors – including secretory breast cancer where it has been recently shown to target WNT signalling (Li et al., Cancer Cell 2007, 12: 542) - and leukemia, but has yet to be characterized in the hematologic setting. Tyrosine receptor kinases (TRK) play key roles in leukemogenesis and already serve as therapeutic targets. We set out to characterize potential downstream targets of ETV6-NTKR3 in AML cells. Methods and Cells By applying molecular cytogenetics, rapid amplification of c-DNA ends, microarray transcriptional profiling, reverse transcriptase quantitative-PCR, sequencing technology, and pathway analysis we defined and characterized the transcriptosome of a t(12;15) cell line (AP-1060) recently established from a patient with acute promyelocytic leukemia. We also investigated the transcriptional responses of AP-1060 cells to TRKi(nhibitor). For comparison we used, firstly a panel of 12 AML cell lines lacking ETV6-NTRK3 or PML-RARA, followed by NB-4 cells with solo PML-RARA. Results FISH confirmed ETV6 rearrangement, while 3′-RACE and RT-PCR identified and confirmed ETV6-NTRK3 fusion transcripts. Sequencing revealed both ETV6 exon-4 / NTKR3 exon-14, and ETV6 exon-2 / exon-18 of NTKR3 (hematopoietic) transcripts - the former dominating. Comparative transcriptional profiling of AP-1060 and control AML cells with or without PML-RARA showed upregulation of RAS-MAPK and PI3K-AKT related genes, highlighting the involvement of both TRK physiological signaling pathways via ETV6-NTRK3. Top genes upregulated in AP-1060 confer signatures both for AML - CCNA1, CD96, DSU, EVI1, HGF, IL32, LGALS3, MDS1, TLE1, TSPAN2; and lymphocyte development - BSPRY, BST1, CCR6, EMP1, GIMAP1, GZMA, PLEKHG1. Several primitive hematopoietic or stem cell mRNAs were also overexpressed, including PRSS2, CD96, SIPA1L2, and PYHIN1. Prominent downregulated genes also included: ADD3, CD36, HOXA-9/10, LGALS9, MALAT1, PGDS, PLA2G4A (AML signature); HOXB4, KIAA1949, NR2F6, TEAD4 (stem cell); and LY6E, TRIM44 (lymphocyte signature). Growth and proliferation of ETV6-NTKR3 cells was exquisitely sensitive to TRKi treatments which spared control AML and to which NB-4 cells were highly resistant. Accordingly we used pharmacologic modulation of conspicuously expressed genes by small molecule TRKi treatment to highlight likely kinase signaling targets among conspicuously expressed genes. Several candidate target genes thus emerged, notably AWNT1, IL32, and the MDS-EVI1 fusion transcript. Salient pharmacologically unmodulated genes were preferentially stem cell in character highlighting this setting for t(12;15) formation in AP-1060 cells. Bioinformatic pathway analysis (http://david.abcc.ncifcrf.gov/) of both up- and down- conspicuously regulated genes identified “Alternative Splicing” as top category, with respectively 743 and 373 alternate spliceform genes up- and down-regulated. These included several genes whose spliceforms may be differentially expressed in oncogenesis, including MDS1-EVI1/EVI1, MALAT1, and WT1/AWT1. Interestingly, a key pre-mRNA splicing gene, MBNL2 was conspicuously downregulated, while another spliceosomal component THOC5 (C22orf19), recently identified as a leukemic kinase signalling target (Pierce et al., Br J Haematol 2008;141:641), is upregulated. Conclusions We present a human leukemia model and resource for ETV6-NTRK3. Taken together, our findings support spliceosomal targeting by ETV6-NTRK3 and suggest a possible underlying mechanistic framework. Additional targets, e.g. WNT signaling, seem to be shared with solid tumors bearing the same oncogene fusion. Perspectives: Future work includes transcriptosomal analysis of AP-1060 cells after knockdown of ETV6-NTRK3 and key splicesomal genes, such as THOC5, by short-hairpin RNAs, and novel, highly selective 4-aminopyrazolylpyrimidine TRKi (Thress et al., Mol Cancer Therapy 2009;8:1818). Disclosures No relevant conflicts of interest to declare.

Description: Abstract 5109 Thymic stromal lymphopoietin (TSLP), a cytokine produced by epithelial cells promotes early B-cell development and activates dendritic cells. It has recently been reported that a subset of B-cell precursor acute lymphoblastic leukemia (pre-B ALL) overexpresses the TSLP receptor CRLF2. CRLF2 overexpression is linked to translocations between sex chromosomes – localizing CRLF2 – and the immunoglobulin heavy chain locus on chromosome 14, or to an interstitial deletion on the gonosomes. Both events, translocation and deletion juxtapose CRLF2 to a different promoter (IgH or P2RY8). Performing quantitative real-time PCR we tested pre-B ALL and acute myeloid leukemia (AML) cell lines for overexpression of CRLF2. AML cell lines were included in the screening because we knew from an earlier TSLP project that the AML cell line MUTZ-3 is TSLP-responsive, and thus positive for the cytokine receptor. Three of 63 (5%) pre-B ALL cell lines tested (INC, MHH-CALL4, MUTZ-5) overexpressed CRLF2 mRNA. CRLF2-high cell lines carry a t(14;Y). With respect to the 58 AML cell lines tested: some expressed CRLF2 mRNA, but none of them rivalled the aforementioned pre-B cell lines. Pre-B ALL cell lines show the association between chromosomal CRLF2 aberrations and JAK2 pseudokinase domain mutations that has been described for primary pre-B ALL cells: cell lines MHH-CALL4 (JAK2I682F) and MUTZ-5 (JAK2R683G) and – newly described - also the CRLF2-high pre-B ALL cell line INC express a mutated version of Janus kinase 2 (JAK2R683G). We established a PCR based assay system that allowed for the rapid detection of the JAK2R683G mutation: none of the CRLF2-low or –negative pre-B ALL cell lines exhibited this mutation. All three CRLF2-high/JAK2mu cell lines showed high phosphorylation levels of the JAK2 downstream target STAT5. Inhibition of the JAK kinase led to dephosphorylation of STAT5. However, repression of 3H-thymidine uptake and induction of apoptosis by inhibition of the JAK2/STAT5 pathway was weaker in the JAK2mu pre-B ALL cell lines than in the JAK2V617F positive essential thrombocythemia-derived cell line SET-2. Provided that these results reflect the situation in primary cells, mutated JAK2 seems to be of lesser importance for growth and survival of pre-B ALL cells than for cells from myeloproliferative neoplasms. The CRLF2-high/JAK2mu cell lines INC, MHH-CALL4 and MUTZ-5 are promising model systems for the study of the roles of high-level CRLF2 expression and of JAK2 mutations in pre-B ALL. Disclosures: No relevant conflicts of interest to declare.

Description: The Philadelphia translocation, encoding the BCR-ABL1 (BCR-ABL) fusion gene, is typically found in chronic myeloid leukemia (CML) and precursor B-cell acute lymphoblastic leukemia (B-ALL), but is exceptionally rare in T-cell acute lymphoblastic leukemia (T-ALL). To study the potential involvement of ABL1 gene rearrangements in T-cell malignancies, we screened 90 T-ALL cases by fluorescence in situ hybridization (FISH), using BCR and ABL1 probes. No BCR-ABL1 fusion signals were observed, confirming the low frequency of this rearrangement in T-ALL, but we did observe marked amplification (〉 10 signals per nucleus) ABL1 of in 5 of 90 (5.5 %) T-ALL patients. Amplification of ABL1 occurred on small extrachromosomal elements that were not detectable by conventional cytogenetics. and hence are referred to as episomes. FISH, and array-CGH analyses delineated the amplicon as a 500 kb region from chromosome band 9q34, containing the oncogenes ABL1 and NUP214 (CAN). Molecular analysis led to the identification of a NUP214-ABL1 fusion gene, which is generated as result of the circularization of the genomic region between ABL1 and NUP214 to form the episomes. This is the first example of an oncogenic fusion gene generated by extrachromosomal amplification. The NUP214-ABL1 transcript was detected in 5 patients with ABL1 amplification, in 5 of 85 (5.8 %) additional T-ALL patients, and in 3 of 22 T-ALL cell lines. The constitutively phosphorylated tyrosine kinase NUP214-ABL1 is sensitive to the tyrosine kinase inhibitor imatinib mesylate (STI-571). The recurrent cryptic NUP214-ABL1 rearrangement is associated with increased expression TLX1 of (HOX11) or TLX3 (HOX11L2), and with deletion of CDKN2A (p16), consistent with a multi-step pathogenesis of T-ALL. Our results identify a novel mechanism for the generation of a fusion gene on extrachromosomal elements, and indicate the importance of activated tyrosine kinase signaling in the pathogenesis of T-ALL. NUP214-ABL1 expression defines a new subgroup of T-ALL patients that could benefit from imatinib treatment.

Description: We recently identified the chimeric kinase FIP1L1-platelet-derived growth factor receptor α (PDGFRα) as a cause of the hypereosinophilic syndrome and of chronic eosinophilic leukemia. To investigate the role of FIP1L1-PDGFRA in the pathogenesis of acute leukemia, we screened 87 leukemia cell lines for the presence of FIP1L1-PDGFRA. One cell line, EOL-1, expressed the FIP1L1-PDGFRA fusion. Three structurally divergent kinase inhibitors—imatinib (STI-571), PKC412, and SU5614—inhibited the growth of EOL-1 cells. These results indicate that the fusion of FIP1L1 to PDGFRA occurs rarely in leukemia cell lines, but they identify EOL-1 as an in vitro model for the study of FIP1L1-PDGFRA-positive chronic eosinophilic leukemia and for the analysis of small molecule inhibitors of FIP1L1-PDGFRα. (Blood. 2004;103:2802-2805)

Description: The methylation of CpG islands leads to the abnormal silencing of tumor suppressor genes and thus supposedly contributes to tumorigenesis. Recently, brain expressed X-linked-2 (BEX2) was described as candidate tumor suppressor gene in malignant glioma. With tissue expression array analyses, we could show that BEX2 was highly expressed in various brain-derived tissues, but not in hematopoetic cells of healthy donors. Also acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) cell lines usually did not show high levels of BEX2. However, we found BEX2 highly expressed in those AML cell lines that carried aberrations of the mixed lineage leukemia gene (MLLmu). While MLL wild-type (MLLwt) cell lines showed hypermethylation of a CpG rich area within the BEX2 promoter, the BEX2 expressing, MLLmu cell lines did not show methylation of the BEX2 promoter. Confirming that BEX2 is an epigenetically regulated gene, aza-2′deoxycytidine increased the expression of BEX2 mRNA in MLLwt cells. Hypermethylation profiles of tumor suppressor genes may be used to identify subtypes of leukemia/lymphoma. To find out whether silencing and activation patterns of BEX2 in leukemia correlate with those of other tumor suppressor genes, we performed a polymerase-chain reaction (PCR) based assay (MLPA) that allowed us to simultaneously verify methylation- and ploidy status of 24 tumor suppressor genes. Twenty-eight human leukemia cell lines were tested, fourteen AML- and ALL-derived cell lines, half of them with MLL translocations. In summary, 7/24 tumor suppressor genes (plus BEX2) appear to be promising markers allowing for the distinction between ALL and AML, and between MLLmu and MLLwt AML. All AML and ALL cell lines were affected by methylation or deletion of CKDN2B and/or ESR1. Analysis of the methylation status of DAPK1 and FHIT allowed to distinguish between ALL and AML. The promoters of both genes, DAPK1 and FHIT were methylated in the majority of ALL cell lines: 11/14 ALL vs. 1/14 AML cell lines showed methylation of DAPK1, 13/14 ALL vs. 2/14 AML showed methylation of FHIT. There was no correlation between the methylation status of tumor suppressor genes and the MLL status in ALL cell lines. In contrast, MLLmu and wt AML cell lines showed differential promoter methylation patterns of IGSF4, RARB and TIMP3. At least two of these genes were methylated in every MLLwt AML cell line, but not in MLLmu AML. Quantitative real-time PCR demonstrated that expression of BEX2 and IGSF4 was silenced in cells carrying the methylated promoters. For RARB and TIMP3, additional mechanisms besides promoter methylation appear to be involved in gene regulation. These results show that methylation status of as few as four tumor suppressor genes (BEX2, IGSF4, RARB and TIMP3) may allow conclusions about the MLLmu/wt status of AML cells. Our results raise the question whether MLLmu proteins are directly or indirectly responsible for the hypomethylation of BEX2, IGSF4, RARB and TIMP3. If this is the case, tissue-specific effectors must play important additional roles as MLLmu ALL cells do not show this phenomenon.

Description: Abstract 4469 The BCR-ABL1 translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). We screened a panel of BCR-ABL1 positive cell lines to find models for imatinib-resistance studies. Five of 19 BCR-ABL1 positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the five resistant cell lines carried mutations in the kinase domain of BCR-ABL1 and – consequently – all also showed resistance to the second generation kinase inhibitors, nilotinib or dasatinib. All Philadelphia chromosome (Ph)-positive cell lines demonstrated constitutive phosphorylation of STAT5 and S6. Imatinib induced dephosphorylation of both BCR-ABL1 downstream effectors in responsive cell lines, but - remarkably – induced dephosphorylation of STAT5 in resistant cell lines as well. By administering well-described signalling pathway inhibitors we were able to show that activation of mTOR complex 1 was responsible for the constitutive S6 phosphorylation of imatinib-resistant cells. Neither BCR-ABL1 nor Src kinases or Ras/Rac-GTPases underlie tyrosine kinase inhibitor resistance in these cell lines. In conclusion, none of the five TKI-resistant cell lines showed aberrant activation of previously-described oncogenic pathways which would explain their resistance. These findings raise the question whether these cell lines might help to find a novel – alternative – explanation for TKI resistance. Interestingly, the proteasome inhibitor bortezomib induced apoptosis in TKI-resistant and –sensitive Ph+ cell lines. Bortezomib is being used for the treatment of multiple myeloma. Our findings support the notion that bortezomib might also be useful for the treatment of imatinib-resistant CML. Disclosures: No relevant conflicts of interest to declare.