BCR-ABL Positive Cell Lines Research Articles

Ferrocene modified analogues of imatinib and nilotinib as potent anti-cancer agents.

The unique features of ferrocene and the need for development of targeted anticancer drugs inspired the design, synthesis and biological evaluation of ferrocenyl modified tyrosine kinase inhibitors by replacing the pyridyl moiety in imatinib and nilotinib generalized structures with a ferrocenyl group. A series of seven new ferrocene analogues were synthesized and evaluated for their anticancer activity in a panel of bcr-abl positive human malignant cell lines using imatinib as a reference drug. The metallocenes exhibited a dose-dependent inhibition on malignant cell growth with varying antileukemic activity. The most potent analogues were compounds 9 and 15a showing comparable or even superior efficacy to the reference. Their cancer selectivity indices suggest a favorable selectivity profile, indicating a 250 times higher preferential activity of 15a towards malignantly transformed K-562 cells and an even twice greater one (500) of 9 in the LAMA-84 leukemic model as compared to the normal murine fibroblast cell line.

Synthesis, molecular modeling, and biological evaluation of novel imatinib derivatives as anticancer agents.

Different derivatives of imatinib were synthesized by a 3-step reaction method. The structures of the new compounds were characterized by spectroscopic methods. For quantitative evaluation of the biological activity of the compounds, MTT assays were performed, where four BCR-ABL negative leukemic cell lines (Jurkat, Reh, Nalm-6 and Molt-4), one BCR-ABL positive cell line (K562), and one non-leukemic cell line (Hek293T) were incubated with various concentrations of the derivatives. Although imatinib was specifically designed for the BCR-ABL protein, our results showed that it was also effective on BCR-ABL negative cell lines except for Reh cell line. Compound 9 showed lowest IC50 values against Nalm-6 cells as 1.639 μM, also the values of Compound 10 for each cell were very close to imatinib. Molecular docking simulations suggest that except for compound 6, the compounds prefer a DFG-out conformation of the ABL kinase domain. Among them, compound 10 has the highest affinity for ABL kinase domain that is close to the affinity of imatinib. The common rings between compound 10 and imatinib adopt exactly the same conformation and same type of interactions in the ATP binding site with the ABL kinase domain.

RUNX1 mutations in blast-phase chronic myeloid leukemia associate with distinct phenotypes, transcriptional profiles, and drug responses

Blast-phase chronic myeloid leukemia (BP-CML) is associated with additional chromosomal aberrations, RUNX1 mutations being one of the most common. Tyrosine kinase inhibitor therapy has only limited efficacy in BP-CML, and characterization of more defined molecular subtypes is warranted in order to design better treatment modalities for this poor prognosis patient group. Using whole-exome and RNA sequencing we demonstrate that PHF6 and BCORL1 mutations, IKZF1 deletions, and AID/RAG-mediated rearrangements are enriched in RUNX1mut BP-CML leading to typical mutational signature. On transcriptional level interferon and TNF signaling were deregulated in primary RUNX1mut CML cells and stem cell and B-lymphoid factors upregulated giving a rise to distinct phenotype. This was accompanied with the sensitivity of RUNX1mut blasts to CD19-CAR T cells in ex vivo assays. High-throughput drug sensitivity and resistance testing revealed leukemia cells from RUNX1mut patients to be highly responsive for mTOR-, BCL2-, and VEGFR inhibitors and glucocorticoids. These findings were further investigated and confirmed in CRISPR/Cas9-edited homozygous RUNX1−/− and heterozygous RUNX1−/mut BCR-ABL positive cell lines. Overall, our study provides insights into the pathogenic role of RUNX1 mutations and highlights personalized targeted therapy and CAR T-cell immunotherapy as potentially promising strategies for treating RUNX1mut BP-CML patients.

PARP Inhibition Sensitize BCR-ABL1 Positive Cel

Introduction: BCR-ABL1 play a key role in the development of chronic myelogenous leukemia and a part of Ph1 positive acute lymphoblastic leukemia (ALL). BCR-ABL1 functions as a tyrosine kinase. Whereas, BCR-ABL1 induces genomic instability by downregulation of BRCA1. An innate error of BRCA1, a molecule involved in the homologous recombination repair pathway, causes hereditary breast and ovarian cancer. PARP inhibitor (PARPi) induces synthetic lethality in BRCA defective cell. Therefore, PARP inhibitor is expected to induce efficient cell death with BCR-ABL1 positive cell. In addition, in some previous reports, reduction of PARP1 activity leads to the upregulation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway and BCR-ABL1 tyrosine kinase activates PI3K/AKT pathway. These findings suggest activation of the PI3K/AKT pathway leading to PARPi resistance in BCR-ABL1 positive leukemic cells. Here, we demonstrate that PARP inhibition attenuates BCR-ABL1 mediated leukemogenesis and aberration of factors associated with PARP inhibitor resistance induces cell death to fully transformed leukemic cells. Method: Bone marrow-derived mononuclear cells (MNC) from wild type mice and BCR-ABL1 transgenic (Tg) mice were exposed to PARPi in vivo, and cell death was analyzed Annexin-V positivity. PARPi sensitivity to BCR-ABL1 expressed cell was also investigated in vivo bone marrow transplantation model using mouse hematopoietic stem cell (HCS) infected with BCR-ABL1 expressing retrovirus. To evaluate more precisely the results obtained in vitro and in vivo transplantation model, the genetical approach was also performed. The Parp1 knockout (KO) mice were crossed with BCR-ABL1 Tg mice. Then, Leukemia development and subsequent mouse death were observed. In vitro, HR activity was examined using DR-GFP assay. Genomic instability was investigated using the breakage-fusion-bridge (BFB) generation.Maintenance of HSC as a progenitor of the leukemic cell was analyzed by repopulation activity using colony assay. The growth-inhibitory effect was assessed using BCR-ABL positive cell lines with PARPi and PI3K inhibitor. Results: BCR-ABL1 Tg mice derived MNC showed more hypersensitivity to PARPi. Mouse HCS was infected with BCR-ABL1 expressing retrovirus and transplanted lethally Olaparib or vehicle was administrated intraperitoneal injection one day after transplantation. BCR-ABL1 mediated leukemic death was observed 1 month after transplantation in sham-treated mouse, whereas, Olaparib treated mouse did not develop BCR-ABL1 mediated leukemia. Parp1 KO BCR-ABL1 Tg mice attenuated leukemia development and extended their survival compared with BCR-ABL1 Tg mice. In vitro experiment revealed HR activity was down-regulated by BCR-ABL1 expression in DR-GFP assay. The number of BFB generation was increased in BCR-ABL1 Tg with Parp1 KO background. The colony-forming activity of BCR-ABL1 positive HSC was totally abolished by PARP inhibition after 3 times serial replating, whereas sham-treated HSC retained repopulation activity. However, the effect of PARPi on BCR-ABL positive leukemic cell lines was controversial. Therefore, leukemic cell lines were treated with the PARPi and inhibitors toward the molecules associated with PARPi resistance. As a result, a combination of PARPi with PI3K inhibitor effectively induce cell death in PARPi resistant BCR-ABL1 positive leukemic cell lines. Conclusion and discussion: Tyrosine kinase inhibitor (TKI) is the gold standard of the therapeutic option of BCR-ABL1 positive leukemia. However, TKI monotherapy is not sufficient for complete eradication of leukemic cells. It is highly expected that molecules effectively induce cell death to leukemic cells combined with TKI. PARPi would be one of these candidates. However, PARPi could not induces efficient death in all of the cancer cells that carry the mutation of molecules associated with the HR defect. Comprehensive genetic analysis to reveal PARPi resistance is important for HRR defective cancer cells. Combination therapy of PARPi and inhibitorstoward the molecules associated with PARPi resistance would be a good therapeutic option for Ph1 positive leukemia. Disclosures No relevant conflicts of interest to declare.

Infliximab therapy together with tyrosine kinase inhibition targets leukemic stem cells in chronic myeloid leukemia

BackgroundExpression of Bcr-Abl in hematopoietic stem cells is sufficient to cause chronic myeloid leukemia (CML) and tyrosine kinase inhibitors (TKI) induce molecular remission in the majority of CML patients. However, the disease driving stem cell population is not fully targeted by TKI therapy, and leukemic stem cells (LSC) capable of re-inducing the disease can persist. Single-cell RNA-sequencing technology recently identified an enriched inflammatory gene signature with TNFα and TGFβ being activated in TKI persisting quiescent LSC. Here, we studied the effects of human TNFα antibody infliximab (IFX), which has been shown to induce anti-inflammatory effects in mice, combined with TKI treatment on LSC function.MethodsWe first performed GSEA-pathway analysis using our microarray data of murine LSK cells (lin−; Sca-1+; c-kit+) from the SCLtTA/Bcr-Abl CML transgenic mouse model. Bcr-Abl positive cell lines were generated by retroviral transduction. Clonogenic potential was assessed by CFU (colony forming unit). CML mice were treated with nilotinib or nilotinib plus infliximab, and serial transplantation experiments were performed.ResultsLikewise to human CML, TNFα signaling was specifically active in murine CML stem cells, and ectopic expression of Bcr-Abl in murine and human progenitor cell lines induced TNFα expression. In vitro exposure to human (IFX) or murine (MP6-XT22) TNFα antibody reduced clonogenic growth of CML cells. Interestingly, TNFα antibody treatment enhanced TKI-induced effects on immature cells in vitro. Additionally, in transplant and serial transplant experiments, using our transgenic CML mouse model, we could subsequently show that IFX therapy boosted TKI-induced effects and further reduced the proportion of malignant stem cells in vivo.ConclusionTNFα signaling is induced in CML stem cells, and anti-inflammatory therapy enhances TKI-induced decline of LSC, confirming that successful targeting of persisting CML stem cells can be enhanced by addressing their malignant microenvironment simultaneously.

Cytotoxic and pro-apoptotic action of MjTX-I, a phospholipase A2 isolated from Bothrops moojeni snake venom, towards leukemic cells

BackgroundChronic myeloid leukemia (CML) is a BCR-ABL1+ myeloproliferative neoplasm marked by increased myeloproliferation and presence of leukemic cells resistant to apoptosis. The current first-line therapy for CML is administration of the tyrosine kinase inhibitors imatinib mesylate, dasatinib or nilotinib. Although effective to treat CML, some patients have become resistant to this therapy, leading to disease progression and death. Thus, the discovery of new compounds to improve CML therapy is still challenging. Here we addressed whether MjTX-I, a phospholipase A2 isolated from Bothrops moojeni snake venom, affects the viability of imatinib mesylate-resistant Bcr-Abl+ cell lines.MethodsWe examined the cytotoxic and pro-apoptotic effect of MjTX-I in K562-S and K562-R Bcr-Abl+ cells and in the non-tumor HEK-293 cell line and peripheral blood mononuclear cells, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and the hypotonic fluorescent solution methods, associated with detection of caspases 3, 8, and 9 activation and poly (ADP-ribose) polymerase (PARP) cleavage. We also analyzed the MjTX-I potential to modulate the expression of apoptosis-related genes in K562-S and K562-R cells.ResultsMjTX-I decreased the viability of K562-S and K562-R cells by 60 to 65%, without affecting the viability of the non-tumor cells, i.e. it exerted selective cytotoxicity towards Bcr-Abl+ cell lines. In leukemic cell lines, the toxin induced apoptosis, activated caspases 3, 8, and 9, cleaved PARP, downregulated expression of the anti-apoptotic gene BCL-2, and upregulated expression of the pro-apoptotic gene BAD.ConclusionThe antitumor effect of MjTX-I is associated with its potential to induce apoptosis and cytotoxicity in Bcr-Abl positive cell lines sensitive and resistant to imatinib mesylate, indicating that MjTX-I is a promising candidate drug to upgrade the CML therapy.

Design, Synthesis, and Biological Evaluation of Novel 1,3,4-Thiadiazole Derivatives as Potential Antitumor Agents against Chronic Myelogenous Leukemia: Striking Effect of Nitrothiazole Moiety.

In an attempt to develop potent antitumor agents, new 1,3,4-thiadiazole derivatives were synthesized and evaluated for their cytotoxic effects on multiple human cancer cell lines, including the K562 chronic myelogenous leukemia cell line that expresses the Bcr-Abl tyrosine kinase. N-(5-Nitrothiazol-2-yl)-2-((5-((4-(trifluoromethyl)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio)acetamide (2) inhibited the Abl protein kinase with an IC50 value of 7.4 µM and showed selective activity against the Bcr-Abl positive K562 cell line. Furthermore, a Bcr-Abl-compound 2 molecular modelling simulation highlighted the anchoring role of the nitrothiazole moiety in bonding and hydrophobic interaction with the key amino acid residues. These results provide promising starting points for further development of novel kinase inhibitors.

Role of ZNF224 in c-Myc repression and imatinib responsiveness in chronic myeloid leukemia.

The transcription factor ZNF224 plays a key proapoptotic role in chronic myelogenous leukemia (CML), by modulating Wilms Tumor protein 1 (WT1) dependent apoptotic genes transcription. Recently, we demonstrated that Bcr-Abl signaling represses ZNF224 expression in Bcr-Abl positive CML cell lines and in CML patients. Interestingly, Imatinib and second-generation tyrosine kinase inhibitors specifically increase ZNF224 expression.On the other hand, Bcr-Abl positively modulates, via JAK2 activation, the expression of the c-Myc oncogene, which is required for Bcr-Abl oncogenic transformation in CML. Consequently, JAK2 inhibitors represent promising molecular therapeutic tools in CML.In this work, we demonstrate that ZNF224 is a novel transcriptional repressor of c-Myc in CML. We also show that ZNF224 induction by Imatinib and AG490, a specific JAK2 inhibitor, is responsible for the transcriptional repression of c-MYC, thus highlighting the crucial role of the ZNF224/c-Myc axis in Imatinib responsiveness.Interestingly, we also report that ZNF224 is induced by AG490 in Imatinib-resistant CML cells, leading to c-Myc repression and apoptosis induction. These findings suggest that the development of molecular tools able to induce ZNF224 expression could provide promising means to bypass Imatinib resistance in CML.

Forced expression of Wnt antagonists sFRP1 and WIF1 sensitizes chronic myeloid leukemia cells to tyrosine kinase inhibitors.

Chronic myeloid leukemia is a clonal myeloproliferative disorder that arises from the neoplastic transformation of the hematopoietic stem cell, in which the Wnt/β-catenin signaling pathway has been demonstrated to play an important role in disease progression. However, the role of Wnt signaling antagonists in therapy resistance and disease progression has not been fully investigated. We aimed to study the effects of Wnt/β-catenin pathway antagonists-secreted frizzled-related protein 1 and Wnt inhibitory factor 1-on resistance toward tyrosine kinase inhibitors in chronic myeloid leukemia. Response to tyrosine kinase inhibitors was analyzed in secreted frizzled-related protein 1 and Wnt inhibitory factor 1 stably transfected K562 cells. Experiments were repeated using a tetracycline-inducible expression system, confirming previous results. In addition, response to tyrosine kinase inhibitor treatment was also analyzed using the secreted frizzled-related protein 1 expressing, BCR-ABL positive MEG01 cell line, in the presence and absence of a secreted frizzled-related protein 1 inhibitor. Our data suggests that total cellular β-catenin levels decrease in the presence of secreted frizzled-related protein 1 and Wnt inhibitory factor 1, and a significant increase in cell death after tyrosine kinase inhibitor treatment is observed. On the contrary, when secreted frizzled-related protein 1 is suppressed, total β-catenin levels increase in the cell and the cells become resistant to tyrosine kinase inhibitors. We suggest that Wnt antagonists carry the potential to be exploited in designing new agents and strategies for the advanced and resistant forms of chronic myeloid leukemia.

Analysis of Copy Number Abnormalities of 86 Acute Lymphoblastic Leukemia Cell Lines Based on the Genetic Subtypes

Introduction: Genetic abnormalities are important to predict prognosis and sometimes can be therapeutic target in pediatric acute lymphoblastic leukemia (ALL). Although the cell lines with recurrent chromosomal abnormalities or leukemic fusions derived from ALL patients are useful tool for various in vitro experiments, it has not been fully investigated whether there is the difference of genetic alterations between clinical samples and cell lines.Here, we performed MLPA analysis of 86 ALL cell lines to determine copy number abnormalities (CNA) and compare with those of the patient's clinical samples.Methods: We performed MLPA analysis of 86 cell lines of ALL (14 with BCR-ABL, 11 with MLL rearrangement, 18 with TCF3-PBX1, 4 with TCF3-HLF, 4 with ETV6-RUNX1 and 35 B-other ALL cell lines) to determine CNA of IKZF1, PAX5, CDKN2A, CDKN2B, ETV6, RB1, BTG1 and EBF1. Then, CNAs were compared to those of patients' samples such as UK cohort (Schwab C, et al. Haematologica, 2013) and Japanese cohort (Asai D, et al. Cancer Med, 2013) according to each specific genetic abnormality, such as BCR-ABL, ETV6-RUNX1, TCF3-PBX1 and MLL-related fusions. In addition, we performed multiplex PCR and RNA-seq to determine fusion transcripts related to Ph-like ALL for the six Ph-negative cell lines with IKZF1 deletions. To determine the expression level of IKZF1 isoform in these cell lines, we performed real time PCR analysis of IKZF1 isoform 1 (IK1) and isoform 6 (IK6).Results: In the BCR-ABL positive cell lines, the frequencies of CDKN2A/2B and BTG1 deletion significantly higher than those in UK cohort (CDKN2A/2B: 100 vs 48%, P <0.01, BTG1; 57 vs 3%, P<0.01). In the TCF3-PBX1 or ETV6-RUNX1 positive group, the frequencies of CDKN2A/2B and BTG1 deletion were increased, but did not obtain statistically significant difference. CNAs of transcriptional factors were not significantly different between cell lines and patient's samples in each genetic subtype. However, IKZF1 deletion tended to increase in BCR-ABL positive (13/14 vs 21/33 in UK cohort, P=0.07) and MLL rearranged cell lines (3/11 vs 2/28 in UK cohort and 0/12 in Japanese cohort, P=0.13 and 0.09). Interestingly, we determined six B other ALL cell lines with IKZF1 deletion. Multiplex PCR analysis determined 5 of six cell lines expressed P2RY8-CRLF2 which was confirmed by Sanger sequence. We also determined high expression of IK1 was observed in half of the BCR-ABL positive cell lines. In contrast, the expression level of IK1 was not increased in the cell lines other than BCR-ABL positive cell lines irrespective of IKZF1 deletion.Discussions: The current analysis revealed the high frequencies of CDKN2A/2B and BTG1 deletion in the BCR-ABL1 positive cell lines compared to those in patents' samples, suggesting that these genetic alterations contributed to the growth advantage. We also determined that CNAs of transcriptional factors were stable in cell lines. Although these findings suggest that CAN of transcriptional factors such as EBF1, PAX5 and ETV6 were less related to growth and survival of leukemic cells, the frequency of IKZF1 deletion tended to be higher in cell lines with BCR-ABL and MLL rearrangement, suggesting IKZF1 deletion might be related to survival of leukemic cells.We also found six B-other cell lines with IKZF1 deletion and determined 5 of the six cell lines expressing P2RY8-CRLF2 fusion. Currently, we are planning comprehensive genetic analysis of these six cell lines using target sequence and RNA-seq. DisclosuresNo relevant conflicts of interest to declare.

Dual HDAC and PI3K Inhibitor, CUDC-907 Alone or in Combination with ABL Tyrosine Kinase Inhibitor Against ABL Tyrosine Kinase Inhibitor Resistant Leukemia Cells

Introduction: Chronic myeloid leukemia (CML) is characterized by the t(9:22) translocation known as the Philadelphia chromosome (Ph). ABL tyrosine kinase inhibitor (TKI), imatinib and second-generation ABL TKIs, nilotinib and dasatinib have demonstrated the potency against CML patients. However, resistance to ABL TKI can develop in CML patients due to BCR-ABL point mutations. Moreover, ABL TKIs do not eliminate the leukemia stem cells (LSCs). Therefore, new approach against BCR-ABL mutant cells and LSCs may improve the outcome of Ph-positive leukemia patients. In eukaryotic cells, histone acetylation/deacetylation is important in transcriptional regulation. Chromatin acetylation is controlled by the opposing effects of two families of enzymes: histone acetyltransferases (HAT) and histone deacetylases (HDACs). Deregulation of HDAC activity may be a cause of malignant disease in humans. Phosphoinositide 3-kinase (PI3K) pathway also regulates cell metabolism, proliferation and survival. Furthermore, aberrant activation of PI3K signaling pathway has been shown to be important in initiation maintenance of human cancers. CUDC-907 is an oral inhibitor of class I PI3K as well as class I and II HDAC enzymes. CUDC-907 is currently being investigated in a pivotal phase 1 clinical trial against hematological malignancies such as malignant lymphoma. We suggested that CUDC-907 mediated inhibition PI3K and HDAC activity and in combination with ABL TKIs may abrogate the proliferation and survival of Ph-positive leukemia cells including T315I mutation and ABL TKI resistant.Materials and methods: In this study, we investigated the combination therapy with a CUDC-907 and an ABL TKIs (imatinib, nilotinib and ponatinib) by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line which was transfected with T315I mutant, nilotinib resistant K562 and ponatinib resistant Ba/F3 cells and primary samples.Results: The treatment of imatinib, nilotinib and ponatinib exhibits cell growth inhibition partially against K562 cells in the presence of feeder cell (HS-5). We found that mRNA of PI3K subunit is significantly increased after a co-culture with HS-5 in K562 and primary CD34 positive CML samples. 72 h treatment of CUDC-907 exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. We examined the intracellular signaling after treatment of CUDC-907. Phosphorylation of JNK, histone acetylation and activity of caspase 3, poly (ADP-ribose) polymerase (PARP) was increased. Anti-apoptotic protein, Mcl-1 was decreased in a dose dependent. We next investigated the efficacy between imatinib and CUDC-907 by using these cell line. Combined treatment of K562 cells with imatinib and CUDC-907 caused significantly more cytotoxicity than each drug alone. Caspase activity was increased and Akt activity was reduced. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved PARP was increased after imatinib and CUDC-907 treatment. We investigated the CUDC-907 activity against T315I positive cells. CUDC-907 potently induced cell growth inhibition of Ba/F3 T315I cells in a dose dependent manner. Combined treatment of Ba/F3 T315I cells with ponatinib and CUDC-907 caused significantly more cytotoxicity than each drug alone. Caspase activity was increased and Akt activity was reduced after ponatinib and CUDC-907 treatment. To assess the activity of ponatinib and CUDC-907, we performed to test on tumor formation in mice. We injected nude mice subcutaneously with Ba/F3 T315I mutant cells. A dose of 20 mg/kg/day p.o of ponatinib and 30 mg/kg/day p.o of CUDC-907 inhibited tumor growth and reduced tumor volume compared with control mice. The treatments were well tolerated with no animal health concerns observed. We also found that the treatment of CUDC-907 exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells, K562 nilotinib resistant cells, T315I mutant primary samples and CD34 positive CML samples.Conclusion: These results indicated that administration of the dual PI3K and HDAC inhibitor, CUDC-907 may be a powerful strategy against ABL TKI resistant cells including T315I mutation and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. DisclosuresNo relevant conflicts of interest to declare.

A Bio-Integrative Approach Identifies an Inflammatory Signature in Chronic Myeloid Leukemia (CML) Stem Cells That Is Highly Perturbed in CML Blast Crisis and Involves REL transcription Factor

Chronic myeloid leukemia is a clonal myeloproliferative neoplasm defined by the presence of BCR-ABL fusion gene. This oncogenic event occurs in a hematopoietic stem cell (HSC) involved in CML initiation, maintenance, relapse and progression. Several evidences suggest that inflammatory pathways may participate to the pathophysiology of CML as well as disease progression to blast crisis. It has been shown that NFKB/REL pathway is constitutively activated both in BCR-ABL positive leukemic cell lines as well as in primary blast cells from CML-BC patients. More recent works identified IL6 as key cytokine acting on CML multipotent progenitors and their normal bystander counterpart to favor their differentiation toward the myeloid lineage. In addition, high levels of autocrine TNFα secretion by quiescent CML stem/progenitor cells activate NFKB pathway and promote their survival. Although all of these observations are linked to inflammatory processes, a focused analysis of inflammatory pathways in primary CML stem cells has not been performed so far. In the present study we undertook a text-mining strategy using pubmed e-querying to generate an exhaustive set of genes linked to inflammation. Then we integrated transcriptome analysis of highly purified CML stem cells to evaluate the contribution of these genes in CML development and progression.Methods : We queried 6 key words (Inflammation, macrophages, inflammatory response, chemokines, leukocytes and interleukins) that returned a total of 332000 hits in Pubmed. A raw gene set of 918 genes was found significantly associated (p<0.05) with these hits. Using R-package, we applied a false discovery rate correction that decreased the set to 588 relevant genes. The expression level of this gene set was then analyzed in previously reported microarray data (GEO accession: GSE47927) of highly purified normal cord blood CD34+CD38-CD90+ HSCs (CB; n=3), chronic phase (CP; n= 6), accelerated phase (AP; n =4) and Blast crisis (BC; n=2) CML cells.Results: Among the 588 genes related to inflammation we found 70 genes differentially expressed between the four groups (normal, CP, AP and BC, p<0.01; ANOVA test). Enrichment analysis confirmed 29 up regulated genes (NES = 2.12; p<0.0001) among which IL-6, PARP1, IL1R2, IRF5, IRF8, IL20. 39 genes such as STAT3, STAT4, CD47, CXCR4 IL-11, IL15, TLR-1, were down-regulated in CML CD34+CD38-CD90+ (all phases) as compared with normal HSCs (NES = -2,58; p<0.0001). Using principal component analysis on the 70 inflammatory deregulated genes we identified 10 genes such as IRAK1, IL1R2, VEGF and ESAM that discriminate "all phase" CML samples from normal HSCs (Dim 2 = 22.7%). Another inflammatory gene subset (n=26 genes) comprising IL6, REL, CXCR4, CXCL2, IL11, TLR1, IL1R2, PPARA highly separated CML stem cells according to the disease phase. The later gene set highly separates CP and AP-CML stem cells from BC-CML stem cell (Dim 1 = 50.3%). We next performed a random forest analysis with machine learning (1000 trees) and found that the inflammatory transcript level that best predicted CML phase was REL transcription factor. The expression of 413 genes were found positively correlated with REL expression in CP, AP and BC-CML CD34+CD38-CD90+ cells (r>0.75 and p-value <0.001). A search using JASPAR and TRANSFAC database identified a significant enrichment of NFKB1 and RELA binding motif in the promoter regions of these 413 genes (p<0.00001) among which several regulatory factors of hematopoietic stem cell biology.Conclusion : Using a bio-integrative approach we identified a specific inflammatory signature in CD34+CD38-CD90+ CML stem cells. This inflammatory network is highly altered in blast crisis suggesting its contribution to disease evolution. We identified REL overexpression as a good predictor for disease progression to blast crisis and found NFKB1and RELA (p=3.2x10-13) as the best REL target candidates. RELA/NFKB1 was previously shown to be constitutively activated in CML and Ph+ ALL and this analysis suggests that this may also take place in the most primitive subset of CML cells although REL may be the main partner of NFKB in CML stem cells. These results which are currently validated using functional assays, could lead to identification of novel therapeutic strategies. DisclosuresTurhan:Bristol Myers Squibb: Consultancy; Novartis: Research Funding.

WT1-mediated repression of the proapoptotic transcription factor ZNF224 is triggered by the BCR-ABL oncogene

The Kruppel-like protein ZNF224 is a co-factor of the Wilms' tumor 1 protein, WT1. We have previously shown that ZNF224 exerts a specific proapoptotic role in chronic myelogenous leukemia (CML) K562 cells and contributes to cytosine arabinoside-induced apoptosis, by modulating WT1-dependent transcription of apoptotic genes. Here we demonstrate that ZNF224 gene expression is down-regulated both in BCR-ABL positive cell lines and in primary CML samples and is restored after imatinib and second generation tyrosine kinase inhibitors treatment. We also show that WT1, whose expression is positively regulated by BCR-ABL, represses transcription of the ZNF224 gene. Finally, we report that ZNF224 is significantly down-regulated in patients with BCR-ABL positive chronic phase-CML showing poor response or resistance to imatinib treatment as compared to high-responder patients. Taken as a whole, our data disclose a novel pathway activated by BCR-ABL that leads to inhibition of apoptosis through the ZNF224 repression. ZNF224 could thus represent a novel promising therapeutic target in CML.

Anti-Leukemic Activity of Phosphoinositide 3-Kinase Inhibitor, Copanlisib in ABL Tyrosine Kinase Resistant Leukemia Cells

Introduction: Chronic myelogenous leukemia (CML) is characterized by cytogenetic aberration (Philadelphia chromosome: Ph) and chimeric oncoprotein BCR-ABL. ABL tyrosine kinase inhibitor (TKI) therapy such as imatinib, nilotinib and dasatinib has improved the survival of Ph-positive leukemia patients. Although impressive clinical responses are obtained in the majority of CML patients, increasing evidence of acquired resistance to TKIs have been documented in clinically. Moreover, ABL TKIs cannot eradicate leukemia stem cells, thus, TKIs do not appear to lead to a cure the diseases. Therefore, new approach against BCR-ABL mutant cells and leukemia stem cells may improve the outcome of Ph-positive leukemia patients. Phosphoinositide 3-kinase (PI3K) pathway regulates cell metabolism, proliferation and survival. Furthermore, aberrant activation of PI3K signaling pathway has been shown to be important in initiation maintenance of human cancers. Copanlisib, also known as BAY80-6946 is a PI3K inhibitor with potential antineoplastic activity. Copanlisib is being investigated in a pivotal phase 2 clinical trial against hematological malignancies such as malignant lymphoma. We hypothesized that targeting PI3K, in combination with ABL TKI, would result in enhanced therapeutic activity in Ph-positive leukemia cells including T315I mutation and ABL TKI resistant.Materials and methods: We investigated the combination therapy with a copanlisib and an ABL TKIs (imatinib, nilotinib and ponatinib) by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line which was transfected with T315I mutant, nilotinib resistant K562, ponatinib resistant Ba/F3 cells and primary sample.Results: 72 h treatment of copanlisib exhibits cell growth inhibition against K562 cells in a dose dependent manner. The treatment of imatinib, nilotinib and ponatinib exhibits cell growth inhibition partially against K562 cells co-cultured with feeder cell line, HS-5. We found that the treatment of copanlisib abrogated the protective effects of HS-5 in K562 cells. We examined the intracellular signaling after treatment of copanlisib. High concentration of copanlisib reduced the phosphorylation of BCR-ABL and Crk-L. Activity of caspase 3 and poly (ADP-ribose) polymerase (PARP) was increased. We next investigated the efficacy between ABL TKI and copanlisib by using these cell line. Phosphorylation of BCR-ABL, Crk-L and Akt was reduced after imatinib and copanlisib treatment. We investigated the copanlisib activity against T315I positive cells. Copanlisib potently induced cell growth inhibition of Ba/F3 T315I cells. Combined treatment of Ba/F3 T315I cells with ponatinib and copanlisib caused significantly more cytotoxicity than each drug alone. Phosphorylation of BCR-ABL and Crk-L was reduced and cleaved PARP was increased after ponatinib and copanlisib treatment. To assess the activity of copanlisib and ponatinib, we performed to test on CML tumor formation in mice. We injected nude mice subcutaneously with Ba/F3 T315I mutant cells. A dose of 20 mg/kg/day p.o of ponatinib and 6 mg/kg/three times per week i.p of copanlisib inhibited tumor growth and reduced tumor volume compared with control mice. The treatments were well tolerated with no animal health concerns observed. We also found that the treatment of copanlisib exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells, K562 nilotinib resistant cells and primary sample.Conclusion: These results indicated that administration of the PI3K inhibitor, copanlisib may be a powerful strategy against ABL TKI resistant cells including T315I mutation and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. DisclosuresOhyashiki:Bristol: Honoraria, Research Funding.

Effect of Imatinib on Proliferation, Apoptosis and Expression of Platelet Derived Growth Factor Receptor By Mesenchymal Stem Cells

IntroductionImatinib Mesylate (IM) is a tyrosine kinase inhibitor (TKI), which targets Platelet Derived Growth Factor Receptor (PDGF-R), c-kit and BCR-ABL and is used in the treatment of Gastrointestinal Stromal Tumors (GIST) and Chronic Myeloid Leukemia (CML). Patients using Imatinib for a long time display abnormalities in bone metabolism (Vandyke et al. J Clin Endocrinol Metab 2013). In vitro, IM affects osteoblastic differentiation through inhibition of PDGF/PDGF-Rb signaling, as well as osteoclast function/differentiation through modulation of PDGF/PDGF-Ra signaling (Berman et al. Leuk Res 2013). Conflicting reports have shown differential effects on in vivo bone marrow density. Confounding factors include patient age (pediatric ve adult), type of bone (osteochondral vs trabecular), duration and dose of IM treatment. Mesenchymal Stem Cells (MSCs) are cells with great regenerative potential and differentiate into adipogenic, osteogenic cells and multiple other cell lineages (Bianco et al. Cell Stem Cell 2008). MSCs express high cell surface levels of PDGF-Rb and intermediate levels of PDGF-Ra and are therefore likely to be affected by IM treatment. Here, we wanted to assess the effect of IM on proliferation and apoptosis of MSCs as well as on surface expression of PDGF-Rb and PDGF-Ra.MethodsHealthy human bone marrow MSCs were isolated using Ficoll and plastic adherence and cultured up till passage 3. Proliferation assays were performed using Real Time Cell Analysis (XCELLigence, Roche) to determine optimal in vitro doses of IM (Novartis). IM was used at doses from 2,5 uM to 20 uM and found to be optimal at 5 uM. PDGF-Rb (CD140b) and PDGF-Ra (CD140a) surface expression were measured using monoclonal antibodies and read using a FACSARIA (Becton Dickinson). Apoptosis was assessed using Annexin-V and Propidium Iodide. Adipogenic and osteogenic differentiation was evaluated after 21 days in differentiation media with or without IM, using spectrophotometric quantitation of levels of Oil Red O (Adipogenic differentiation) and Calcium Phosphate (Osteogenic differentiation).ResultsIM inhibited proliferation of MSCs in a dose-dependent fashion, with doses > 5 uM resulting in severe suppression of proliferation. Inhibition of proliferation by IM could be overcome by increasing cell densities of MSCs, but not by addition of PDGF-BB. Co-treatment with IM and PDGF-BB resulted in more pronounced suppression of MSC proliferation. Treatment with IM resulted in a decrease of cell surface expression of both PDGF-Rb from 97,4±2,36% to 77,3±0,13% (n=3, p<0.02) and PDGF-Ra from 18,4% to 6,4% (n=1). Addition of PDGF-BB resulted in a further decrease in cell surface expression of PDGF-Rb, but had no effect on expression of PDGF-Ra. IM increased apoptosis levels (Annexin-V positive cells) about twofold. Addition of 5 or 10 ng/mL PDGF could completely abrogate this effect. Treatment of K562, a BCR-ABL positive CML cell line, with 5 uM IM suppressed proliferation of K562 four-fold, but had no obvious effect on levels of apoptosis. Treatment of MSCs with IM during differentiation revealed no clear effect on adipogenesis, but did increase osteogenic differentiation, as measured as an increase in Calcium-Phosphate.ConclusionsImatinib Mesylate inhibits PDGF/PDGF-R signaling through interference with tyrosine kinases. Here, we found that 5 uM IM not only affects MSC proliferation through inhibition of PDGF-receptor signaling, but also through downregulation of PDGF-Rb and PDGF-Ra cell surface expression. This dose is very close to the maximal plasma concentration of 4.6 uM observed in patients (Druker et al. New England J Med 2001). Addition of PDGF-BB enhanced the effects of IM on suppression of MSC proliferation, likely through further downregulation of surface PDGF-Rb expression, thus decreasing PDGF/PDGF-R signaling. PDGF signaling has been implicated in regulation of invasiveness of cancers and TKI have been used in the treatment of several types of cancer. Whether or not combination treatment of IM with PDGF-BB similarly affects proliferation/invasiveness of BCR-ABL+ CML cells, and PDGF-Ra positive GIST cells, remains to be investigated. Prolonged treatment with IM in patients has been shown to affect bone remodeling and bone densities. Our current results suggest that treatment with IM impacts BM-resident MSCs, supporting increased osteogenesis. DisclosuresNo relevant conflicts of interest to declare.

Synthesis and kinase inhibitory activity of new sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazines

A new series of sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazine has been synthesized and characterized. Their anticancer activity was tested in vitro against multiple human cancer cell lines and were found to have dose-dependent antiproliferative effects. Furthermore, some of the new compounds inhibited the Abl protein kinase with low micromolar IC50 values and exhibited selective activity against the Bcr-Abl positive K562 and BV173 cell lines, providing starting points for the further development of this new kinase inhibitor scaffold.

Inhibition Of Ectodomain Shedding Of Rptp-κ Rescues TKI-Resistance In BCR-ABL Positive Leukemia Through Down-Regulating β-Catenin Transcriptional Activity

BackgroundTyrosine kinase inhibitor (TKI) resistance is a major challenge in treatment of BCR-ABL positive leukemia such as chronic myeloid leukemia (CML). The latest studies have demonstrated that super-activation of β-catenin plays pivotal roles in TKI resistance. RPTPκ, a receptor protein-tyrosine phosphatase, functions as a tumor suppressor by down regulating β-catenin activity when expressed as an integral receptor on plasma membrane, while release of its intracellular domain PIC after two-step-cleavage by ADAM 10 and γ-secretase will up-regulate the transcriptional activity of β-catenin. Objectives and methodsHypothesizing that down-regulation of PIC level via inhibition of RPTPκ shedding might regulate TKI resistance of BCR-ABL positive leukemia cells by decreasing the activity of β-catenin and inhibiting the proliferation of leukemia cells, we aim in this study to identify PIC and β-catenin expression level in TKI sensitive or resistant BCR-ABL positive leukemia cells, and the correlation between their transcriptional activities and cell proliferation rate. To provide theoretical support for the development of a potent RPTPκ-targeting drug with low toxicity, we've produced a rabbit polyclonal antibody specifically targeting the cleavage site of ADAM 10 on RPTPκ, and determined its efficacy and specificity on regulating β-catenin's activity and TKI resistance in vitro. ResultsBy using qRT-PCR and Western blot, we have found that in TKI resistant cell line and CML patients, levels of RPTPκ expression, intracellular shedding product PIC and activated form of β-catenin are all significantly higher than those in TKI-sensitive cell line or CML patients. To confirm the role of β-catenin activity and RPTPκ shedding in TKI resistance, we artificially up-regulated β-catenin activity by treating the BCR-ABL positive cell line KU812 with GSK 3β inhibitor treatment. We found that KU812, which used to be sensitive to TKI, became resistant to TKI upon such treatment, and such resistance could be reversed by inhibiting RPTPκ shedding with γ-secretase inhibitor (GSI). Furthermore, overexpression of PIC by lentivirus infection in KU812 also induced its resistance to TKI. Similarly, the TKI resistant BCR-ABL positive cell line Sup-B15 became sensitive to TKI after GSI treatment, in the light of its cell proliferation reduced by at least 40%, suggesting that RPTPκ shedding might play a pivotal role in TKI resistance of these cell lines. Given the broad substrates of γ-secretase, we developed a polyclonal antibody against ADAM10 cleavage site on RPTPκ to inhibit its shedding, and found that the polyconal antibody could effectively rescue TKI resistance of Sup-B15. ConclusionOur results confirmed the super-activation of β-catenin played pivotal roles in TKI resistance, and demonstrated for the first time the role of RPTPκ for β-catenin activity and TKI resistance in BCR-ABL positive cell lines, contributing to the following targeted drug development and refractory BCR-ABL positive leukemia management. Disclosures:No relevant conflicts of interest to declare.

Role Of Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) and Its Ligands In The Regulation Of Functional OCT-1 Activity In CML Cells

IntroductionThe human organic cation transporter-1 (hOCT-1) is the primary active influx protein for imatinib in BCR-ABL positive cells. The functional activity of the OCT-1 protein (OCT-1 activity, OA) is predictive of molecular response in de-novo chronic phase chronic myeloid leukemia (CP-CML) patients. We have previously demonstrated that diclofenac, a competitive peroxisome proliferator-activated receptor-γ (PPARγ) antagonist, can significantly increase OA in CML cells 1. However, the role of PPARγ and its ligands in OA regulation remain unknown. Thus, the link between OA and PPARγ in CML cells has been investigated in this study. MethodsOA was determined by intracellular uptake and retention assay (IUR) in the presence and absence of the OCT-1 inhibitor, prazosin 2. To assess the effect of PPARγ ligands on OA, BCR-ABL positive cell lines (KU812, K562) were incubated with PPARγ antagonist (GW9662, T0070907) or agonists (GW1929, rosiglitazone) respectively for 1 hour immediately prior to the IUR assays. The OA was also assessed in the mononuclear cells (MNCs) of 77 CP-CML patients enrolled to the TIDEL II trial. PPARγ activity in CML MNC nuclear extracts was determined through the use of a PPARγ Transcription Factor Assay Kits according to the manufacturer's instructions. To assess the effect of PPARγ ligands on cell death, KU812 or K562 cells were stained with AnnexinV and 7-AAD for detection of apoptosis after the co-administration of imatinib and PPARγ ligands for 72 hours. ResultsA significant increase in OA was observed in KU812 and K562 cells treated with PPARγ antagonists. In contrast, PPARγ agonists significantly decreased the OA in both cell lines (Table 1). A negative link between OA and PPARγ activity was observed in CML MNC samples (R=-0.585, p<0.001). PPARγ activity was significantly elevated in CML patients who had a low OA at diagnosis (less than 4 ng/200,000 cells) compared with those who had higher OA (p<0.001). After 72 hours co-administration with 0.1µM imatinib, KU812 cells treated with PPARγ antagonists (GW9662 and T0070907) showed a significantly lower cell viability (40% and 18% respectively) compared with vehicle control (70%, p<0.001). Similar results were also observed in K562 cells after co-administration with 1.0µM imatinib for 72 hours. K562 cells treated with PPARγ antagonists (GW9662: 51% and T0070907: 47%) showed a significantly lower cell viability (51% and 47% respectively) compared with vehicle control (61%, p<0.05).Table 1The effect of PPARγ ligands on OCT-1 activity (percentage of vehicle control, *p<0.05)PPARγ antagonistsPPARγ agonistsGW9662T0070907GW1929RosiglitazoneKU812 (n=6)121.3%*130.3%*71.4%*52.4%*K562 (n=3)124.8%*127.7%*66.5%*56.2%* ConclusionLigand-activation or inhibition of PPARγ is a regulator of OA in CML cell lines, and the low MNC OCT-1 activity in CML patients is consistent with the high level of PPARγ activity in these cells. Low PPARγ activity may be the key driver for low OA and poor imatinib response observed in a subset of CML patients. Importantly, the enhanced OA as a result of PPARγ antagonist treatment resulted in increased cell death following co-administration with imatinib. Ongoing studies relating to the upstream pathways involved in PPARγ activation aim to reveal the possible mechanism of OA modulation by PPARγ. Enhancement of OA by PPARg antagonists is likely to provide an important axis for clinical application to improve the clinical efficacy of imatinib. This would be particularly important in patients with low OA who currently have inferior outcomes with imatinib therapy.1. Wang J, Hughes TP, Kok CH, et al. Contrasting effects of diclofenac and ibuprofen on active imatinib uptake into leukaemic cells. British Journal of Cancer. 2012;106(11):1772-1778. 2. White DL, Saunders VA, Dang P, et al. Most CML patients who have a suboptimal response to imatinib have low OCT-1 activity: Higher doses of imatinib may overcome the negative impact of low OCT-1 activity. Blood. 2007;110(12):4064-4072. Disclosures:Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL: Research Funding. White:Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding.

Efficacy Of The Polo-Like Kinase Inhibitor, Rigosertib Alone Or In Combination With ABL Tyrosine Kinase Inhibitor Against BCR-ABL-Positive Leukemia Cells

Chronic myeloid leukemia (CML) is characterized by the t(9:22) translocation known as the Philadelphia chromosome (Ph). ABL tyrosine kinase inhibitor (TKI), imatinib and second-generation ABL TKIs, nilotinib and dasatinib have demonstrated the potency against CML patients. However, resistance to ABL TKI can develop in CML patients due to BCR-ABL point mutations. Moreover, ABL TKIs do not eliminate the leukemia stem cells (LSCs). These leukemia cells are contained within a niche in the bone marrow and are often impervious to current treatments. Therefore, new approach against BCR-ABL mutant cells and LSCs may improve the outcome of Ph-positive leukemia patients. Polo-like kinases (PLK) are the family of serine threonine kinases and essential for mitosis. PLK is also critical regulator of cell cycle progression and DNA damage response. One of the PLK and phosphoinositide 3-kinase (PI3K) inhibitor, rigosertib (ON 01910.Na) is a novel synthetic benzyl styryl sulfone that is cytotoxic against a variety of human tumor cell lines. Rigosertib is currently being investigated in a pivotal phase 3 clinical trial against hematological malignancies such as myelodysplastic syndromes (MDS). We suggested that rigosertib mediated inhibition PLK and PI3K activity and in combination with ABL TKIs may abrogate the proliferation and survival of Ph-positive leukemia cells including T315I mutation and ABL TKI resistant. In this study, we investigated the combination therapy with a rigosertib and an ABL TKIs (imatinib, nilotinib and ponatinib) by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line with T315I mutant, nilotinib resistant K562 and ponatinib resistant Ba/F3 cells. 72 h treatment of rigosertib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. The treatment of imatinib, nilotinib and ponatinib exhibits cell growth inhibition partially against K562 cells in the presence of feeder cell (HS-5) conditioned media. We found that the treatment of rigosertib abrogated the protective effects of HS-5 conditioned media in K562 cells. We examined the intracellular signaling after treatment of rigosertib. Phosphorylation of BCR-ABL, Crk-L was not reduced. However, activity of caspase 3 was increased. We next investigated the efficacy between imatinib and rigosertib by using these cell line. Combined treatment of K562 cells with imatinib and rigosertib caused significantly more cytotoxicity than each drug alone. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved PARP and γH2A.X phosphorylation was increased after imatinib and rigosertib treatment. Anti-apoptotic protein, Mcl-1 was also decreased. We also found the phosphorylation of histone H3 was increased after rigosertib treatment suggested that the cells arrested in G2/M phase. We investigated the rigosertib activity against T315I positive cells. Rigosertib potently induced cell growth inhibition of Ba/F3 T315I cells. Combined treatment of Ba/F3 T315I cells with ponatinib and rigosertib caused significantly more cytotoxicity than each drug alone.We next investigated by using ponatinib resistant Ba/F3 cells and nilotinib resistant K562 cells. In the ponatinib resistant cell lines, IC50 of ponatinib was up to 200 nM. BCR-ABL triple point mutations (T315I, E255K and Y253H) were detected by direct sequence analysis. The treatment of rigosertib exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells and K562 nilotinib resistant cells. These results indicated that administration of the PLK and PI3K inhibitor, rigosertib may be a powerful strategy against ABL TKI resistant cells and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. Disclosures:Ohyashiki:Novartis: Honoraria, Research Funding.

The Role Of SOCS1 In BCR-ABL Mediated Transformation and Leukemogenesis

The BCR-ABL oncogene activates several signaling pathways, most notably by constitutive phosphorylation of the signal transducer and activator of transcription protein 5 (STAT5). After phosphorylation and nuclear translocation, STAT5 transcriptionally activates numerous genes responsible for proliferation, survival and differentiation of hematopoietic stem and progenitor cells. Among the STAT5 target genes are suppressor of cytokine signaling (SOCS) proteins. SOCS proteins inhibit JAK kinases by multiple mechanisms, thereby terminating cytokine signaling in a classical negative feedback loop. The SOCS family of proteins comprises eight members: cytokine-inducible SRC homology 2 (SH2) domain protein (CIS) and SOCS1–SOCS7. SOCS1 is frequently silenced by hypermethylation in multiple myeloma and inactivating mutations have been found in Hodgkin lymphoma with consecutive increase in JAK2 kinase activity. More recently, we identified SOCS1 as a “conditional oncogene” in the context of the FLT3-ITD oncogene (Reddy et al, Blood 2012): SOCS1 significantly enhanced FLT3-ITD-mediated myeloid transformation, both in vitro and in vivo. We hypothesized that this may be a more general mechanism of transformation and therefore analyzed the role of SOCS proteins in BCR-ABL mediated transformation and leukemogenesis.First, we investigated gene expression levels of SOCS proteins in BCR-ABL positive (versus BCR-ABL negative) cell lines and primary ALL long term-cultured cells. Upon treatment with the BCR-ABL inhibitor imatinib, mRNA expression levels of CIS and SOCS1-4 were reduced. SOCS5-7 did not exhibit any changes and were non-responsive to ABL-kinase inhibition. In lineage-depleted primary murine bone marrow retrovirally transduced with BCR-ABL, high induction of CIS and SOCS1-3 mRNA was detected, while SOCS4-7 showed only minor changes. When overexpressed in IL-3 dependent cell lines, SOCS1 led to a very rapid cell death within few days. Similar effects were demonstrated for CIS and SOCS2 overexpression, however, with a slower kinetics. In contrast, BCR-ABL transduced cells were insensitive to SOCS1 overexpression. In colony formation assays performed with primary hematopoietic cells, expression of SOCS1 led to a significant decrease of colony numbers. Interestingly, co-expression of SOCS1 and BCR-ABL (hereafter abbreviated as SOCS1/BCR-ABL) also lowered colony numbers compared to cells expressing BCR-ABL alone. However, when cells were subjected to interferon alpha or interferon gamma treatment, SOCS1/BCR-ABL positive cells displayed higher colony numbers and gained a growth advantage over BCR-ABL expressing cells, since anti-proliferative effects of the cytokines were inhibited by the presence of SOCS1. A careful analysis of the downstream signaling cascade of BCR-ABL and SOCS1/BCR-ABL expressing cells did not demonstrate any differences in the phosphorylation of AKT, ERK1/2 and STAT5. However, when BCR-ABL was inhibited by imatinib, STAT5 phosphorylation was significantly decreased in SOCS1/BCR-ABL transduced cells. Finally, the influence of SOCS1 in BCR-ABL mediated leukemia was investigated in a murine bone marrow transplantation model. BCR-ABL or SOCS1/BCR-ABL expressing cells led to disease formation with a chronic myeloid leukemia-like phenotype. Interestingly, the co-expression of SOCS1 and BCR-ABL prolonged disease latency, as opposed to the phenotype observed with FLT3-ITD (where SOCS1 co-expression shortened latency). In this setting SOCS1 acts as a tumor suppressor, protecting BCR-ABL transformed cells from rapid disease development, and a molecular analysis will be presented. Disclosures:No relevant conflicts of interest to declare.