ALBERT

Description: Chronic myelogenous leukemia (CML) invariably progresses to blast crisis, which represents the most proliferative phase of the disease. The BCR-ABL1 oncogene stimulates growth and survival pathways by phosphorylating numerous substrates, including various Src family members. Here we describe up-regulation, in contrast to activation, of the ubiquitously expressed Src kinase, Fyn, by BCR-ABL1. In a tissue microarray, Fyn expression was significantly increased in CML blast crisis compared with chronic phase. Cells overexpressing BCR-ABL1 in vitro and in vivo display an up-regulation of Fyn protein and mRNA. Knockdown of Fyn with shRNA slows leukemia cell growth, inhibits clonogenicity, and leads to increased sensitivity to imatinib, indicating that Fyn mediates CML cell proliferation. In severe combined immunodeficient (SCID) mice injected with Fyn shRNA–expressing cells, myeloid-derived cell numbers dropped by 50% and death from leukemia was delayed. Taken together, these results encourage the development of therapies targeting Fyn expression.

Description: Development and progression of leukemia requires interaction of leukemia-initiating cells with their bone marrow niches. The niches serve as the nursery and shelter for the leukemic cells, which can result in drug resistance, disease recurrence, and minimal residual disease, the most important causes for the death of patients with leukemia. Therefore, obliteration of the interaction between the leukemic cells and their niches is of utmost importance in eradicating leukemic cells during therapy to cure the disease. However, little is currently known of the molecular mechanisms underlying the interaction of the two types of cells. Sox4, a SRY-related HMG-box containing transcription factor that is vital during development, plays an important role in leukemia. Published mouse studies demonstrated that increased expression of Sox4 was associated with leukemogenesis. We determined the expression levels of Sox4 by real-time RT-PCR in 100 human leukemic samples and found high levels of expression in B- and T-ALL, but not in AML, CML, CLL, Sezary syndrome, or T cell prolymphocytic leukemia. In accordance, 7 of the 8 ALL cell lines (the exception was 697) we tested showed high expression levels of Sox4, but AML cell lines, normal mature B cells, T cells, and bone marrow CD34+ cells had low levels of expression. Since the majority of clinical B-ALL cases correspond to the pre-B cell stage, we investigated the role of Sox4 in a pre-B cell line (Nalm6) by lentivirus-mediated RNAi. Remarkably, knockdown of Sox4 in Nalm6 cells caused 70% reduction in the formation of leukemic cell clusters under the monolayer of co-cultured M2-10B4 bone marrow stromal cells, a phenomenon known as pseudo-emperipolesis. Similar results were obtained with ex vivo cultured bone marrow cells from conditional Sox4 knockout mice that displayed B cell developmental arrest at the transition from pro-B to pre-B cell stage and an absence of pre-B cells. These findings suggested that Sox4 is required for the interaction of the developing B cells or leukemic cells with bone marrow stromal cells, a component of the bone marrow niche. Since CXCR4/SDF1-mediated “homing” is known to be required for pseudo-emperipolesis, we tested the effect of Sox4 on Nalm6 cell migration toward SDF1 gradient and found that Sox4 did not affect the migration, suggesting that Sox4 is not acting through “homing”. Instead, our data indicated that the role of Sox4 in the interaction of leukemic cells with stromal cells is most likely mediated by its ability in enhancing the adhesion of the leukemic cells because we found that lentivirus-medicated overexpression of Sox4 in the 697 B cell line caused the suspension cells to display a spindle and adhesive morphology. In addition, 21% of the putative Sox4 downstream genes that we identified by multiple sets of gene expression microarray experiments are known to be involved in cell adhesion. Moreover, we found that the changes in gene expression profile of leukemic cells upon Sox4 knockdown or overexpression significantly overlap with the changes in response to the presence of bone marrow stromal cells in co-culture, indicating that Sox4 pathways are involved in leukemic cell response to stromal cell signaling. Based on these findings we hypothesize that deletion of Sox4 abolishes the interaction between the developing lymphocytes and their niches during lymphopoiesis. Conversely, overexpression of Sox4 may enforce these cells to over-interact with the niches so that they are overexposed to local growth factor stimuli. If superimposed with other genetic and/or epigenetic changes in the developing lymphocytes, such over-interaction may result in the development of leukemia. In case of established leukemia, such over-interaction may lead to the enhanced protection of leukemic cells by their niches. Therefore, the role of Sox4 in the interaction of developing lymphocytes or leukemic cells with their niches is like “rooting into the soil” of a growing tree, abbreviated as “rooting”.

Description: Bcr-Abl is a leukemia-inducing protein, which causes oncogenic transformation of myeloid progenitors in Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML) and lymphoid progenitors in Ph+ acute lymphoid leukemia (ALL). Oncogenic transformation of hematopoietic cells by the Bcr-Abl oncoprotein directly involves the activation Jak2 tyrosine kinase and the Stat5 transcription factor. Both proteins are normally linked to the IL-3/GM-CSF receptors for growth and survival. Since fibroblastic cells are not targets of BCR-ABL induced oncogenesis, we determined whether forced expression of the IL-3 receptor would allow oncogenic transformation of NIH 3T3 fibroblasts known to be resistant to transformation by BCR-ABL. NIH 3T3 cells transduced with the human IL-3 receptor a and b chains were highly susceptible to oncogenic transformation by expression of BCR-ABL. Forced expression of both receptor chains but not either one alone allowed efficient foci formation of NIH 3T3 cells expressing BCR-ABL, and these cells formed colonies in soft agar whereas BCR-ABL positive NIH 3T3 cells lacking IL-3 receptor expression did not. The Bcr-Abl kinase inhibitor imatinib mesylate (1 mM) and the Jak kinase inhibitor AG490 (10 mM) strongly inhibited agar colony formation. A small molecule inhibitor of Jak2 kinase, 1,2,3,4,5,6-hexabromocyclohexane reported to be specific for Jak2 (Sandberg et al. J. Med. Chem, 2005)-significantly reduced the phosphorylation of Gab2 at the YxxM motif, which is needed for activation of the PI-3 kinase and Akt, two proteins that are part of the Bcr-Abl/Jak2 Network (Samanta et al. Cancer Res, 2006). These findings indicate that Bcr-Abl oncoprotein requires the IL-3 receptor/Jak2/Stat5 pathways for oncogenic transformation of NIH 3T3 fibroblasts, and may explain partially why Bcr-Abl oncogenesis is restricted to hematopoietic malignancies. Furthermore, this cell system in fibroblastic and other cell lineages will provide a model to probe the detailed steps that require IL-3 receptor and Jak2 for Bcr-Abl induced leukemia.

Description: Imatinib mesylate is effective against Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL) but, when used as a single agent, responses are transient and most patients relapse within 4–6 months. AMN107 is a novel oral aminopyrimidine ATP-competitive inhibitor of the protein tyrosine kinase activity of Bcr-Abl. Following oral administration to animals, AMN107 is well absorbed, has a good pharmacokinetic profile, and is well tolerated. The activity of AMN107, relative to imatinib, in both Ph-positive (Z-119 and Z-181) and Ph-negative (Z-138) ALL cell lines was studied. Z-119 and Z-181 cells were derived from Ph-positive ALL patients and retained typical B-cell characteristics and phenotypes of the original leukemia, including cytogenetic abnormality t(9;22) and p190 Bcr/Abl kinase. Z-138, a Ph-negative cell line, was derived from a patient with chronic lymphocytic leukemia and supervening ALL. Treatment with AMN107 or imatinib for 3 days (MTS assay) inhibited proliferation of Z-119 cells with the IC50 values of 19.3 nM and 620.0 nM, respectively, revealing AMN107 to be 32 fold more potent than imatinib. Treatment of Z-181 cell line lasted for 4 days (MTS assay) because of lower growth rate of these cells: IC50 for AMN107 and imatinib were 1.6 nM and 63.9 nM, respectively, showing AMN107 to be 40 fold more potent than imatinib. Neither drug showed activity against Ph-negative Z-138 cells. We also compared the activity of AMN107 in Ph-positive ALL cell lines expressing p190 Bcr/Abl protein to that in Ph-positive chronic myeloid leukemia cell lines KBM5 and KBM7 expressing p210 Bcr/Abl protein. The activity was similar with IC50 in KBM5 cells of 11.3 nM and in KBM7 cells of 4.3 nM. In experiments focused on cell cycle analysis we found that at equipotent doses (as determined by MTS assay) both drugs induced cell accumulation in G0/G1 phase in Z-119 but not in Z-181. We demonstrated that increasing equipotent concentrations of AMN107 and imatinib induced activation of caspase-3 that resulted in apoptosis, as assessed by propidium iodide staining, in Z-119 cells, while Z-181 cells showed lack of apoptotic response. Following treatment with a broad range of AMN107 and imatinib doses for 3 hrs, Bcr/Abl expression and phosphorylation were determined in Z-119 cells by immunoprecipitation and Western blotting: Bcr/Abl phosphorylation was inhibited completely with AMN107 at 125.0 nM, and with imatinib at 2500 nM, confirming again the higher potency of AMN107. Finally, similar differential effect of AMN107 and imatinib on Bcr/Abl protein expression and phosphorylation was observed in leukemic cells obtained from blood of Ph-positive ALL patients. We conclude that AMN107 has significant activity against Ph-positive ALL cells and warrants investigation in patients with Ph-positive ALL.

Description: Abstract 515 The success of tyrosine kinase inhibitors as first line therapy for t(9;22) Chronic Myelogenous Leukemia (CML) depends on the addiction that Philadelphia-positive (Ph+) hematopoietic progenitors, but not quiescent Ph+ stem cells, have for BCR-ABL1 tyrosine kinase activity. We reported that the activity of the tumor suppressor PP2A is inhibited in a SET-dependent manner in CML progenitors and CD34+/CD38- stem cells from chronic phase (CP) and, to a greater extent, blast crisis (BC) CML patients (Neviani P. et al.: Cancer Cell 2005, J. Clin. Invest 2007, and ASH 2008). Restoration of PP2A activity by the immunosuppressive sphingosine analogue FTY720 markedly decreases the number of Ph+ but not normal long-term culture-initiating cells (LTC-IC) and quiescent stem cells (CFSEMAX/CD34+) by suppressing the BCR/ABL kinase-independent enhancement of b-catenin expression/transcriptional activity (Oaks JJ., et al., ASH 2009). Here we report that FTY720 induces apoptosis of Ph+ CD34+/CD38- cells independent from its phosphorylation as treatment with a phosphorylated FTY720 did not alter the number of Ph+ CFSEMAX/CD34+ cells. By contrast, two non phosphorylatable and non immunosuppressive FTY720 derivatives did significantly affect survival of Ph+ stem/progenitor cells. Interestingly, we also noted that the activity but not expression of BCR-ABL1 is considerably lower in quiescent CFSEMAX/CD34+ than CFSE+/CD34+ cells that underwent at least one division (∼80% decrease; n=3). Conversely, BCR-ABL1 expression is significantly higher in quiescent than proliferating CFSE+/CD34+ cells, suggesting that BCR-ABL1 might serve as a scaffold for other kinase(s) able to sustain survival and quiescence of Ph+ stem cells. Indeed, we found that expression of the K1172R kinase-deficient BCR-ABL1 mutant enhances expression and activity of Jak2, a kinase that is not only associated with BCR-ABL1 but is also capable of inactivating and being inactivated by PP2A. Accordingly, lentiviral shRNA-mediated BCR-ABL1 downregulation in Ph+ CD34+/CD38- stem cells resulted in marked (≥70% inhibition, P

Description: Abstract 4181 Ahi-1 (Abelson helper integration site-1) is an oncogene that was initially identified by provirus insertional mutagenesis in v-abl-induced murine pre-B cell lymphoma. The Ahi-1/AHI-1 protein contains an SH3 domain, multiple SH3 binding sites and a WD40-repeat domain, all known to be important mediators of protein-protein interactions. Human AHI-1 is highly deregulated in human leukemic cells, particularly in BCR-ABL+ leukemic stem cells from patients with chronic myeloid leukemia (CML). We have demonstrated that overexpression of Ahi-1 in primitive hematopoietic cells confers a growth advantage in vitro and induces leukemia in vivo; these effects can be enhanced by BCR-ABL, a fusion oncogene that plays a major role in the genesis of CML. AHI-1 can physically interact with BCR-ABL and JAK2 in CML cells and this interaction complex further mediates tyrosine kinase inhibitor (TKI) response/resistance of CML stem/progenitor cells. Despite its importance, the mechanism by which this complex affects cell proliferation and survival and regulates sensitivity of CML cells to TKIs remains unknown. To identify and characterize which functional domain(s) of Ahi-1 is critical for its interaction with BCR-ABL and/or Jak2, full length Ahi-1 and several mutant forms, including N-terminal deletion (N-terΔ, containing both SH3 and WD40-repeat domains), SH3 deletion (SH3Δ) and double WD40-repeat domain and SH3 domain deletions (SH3WD40Δ) were generated and stably transduced into BCR-ABL inducible BaF3 cells, in which the level of expression of BCR-ABL can be down-regulated by exposure to doxycycline. Epitope-tagged full length and mutant Ahi-1 constructs were also transiently expressed in 293T cells co-expressed with either BCR-ABL or Jak2. Co-IP experiments showed that Ahi-1 is highly expressed and stably associated with BCR-ABL-Jak2 complex in BCR-ABL inducible cells co-transduced with full-length Ahi-1 and that it can directly interact with Jak2; Ahi-1 was detected in both Ahi-1-transduced BaF3 cells (without BCR-ABL) and BCR-ABL inducible cells after IP with a Jak2 antibody. Interestingly, N-terminal region of Ahi-1 was required for Ahi-1-Jak2 interaction as a predicted 70 kD product was not detectable in the same cells transduced with the Ahi-1N-terΔ mutant. In contrast, N-terminal region of Ahi-1 was not associated with Ahi-1-BCR-ABL interaction since BCR-ABL was still detectable in BCR-ABL inducible cells co-transduced with the Ahi-1N-terΔ mutant after IP with the anti-ABL antibody. The deletion of either the SH3 domain or both the SH3 and WD40-repeat domains did not interfere with their interaction with Jak2. Ahi-1 lacking both the SH3 and WD40-repeat domains lost its ability to interact with BCR-ABL when the SH3WD40Δ mutant and BCR-ABL were co-expressed in 293T cells and the SH3 deletion did not affect Ahi-1's interaction with BCR-ABL, indicating that the WD40-repeart domain is required for a direct Ahi-1-BCR-ABL interaction. As expected, overexpression of full-length Ahi-1 in BCR-ABL inducible cells resulted in fewer Annexin V+ apoptotic cells after imatinib (IM) treatment compared to BCR-ABL inducible cells (5.9% and 8% v.s.26% and 34% with either 2 or 5 μM IM) after 24 hours. Interestingly, cells expressing the SH3WD40Δ mutant displayed dramatically increased cell sensitivity to IM with increased Annexin V+ cells (62% and 69% v.s.5.9% and 8%), while cells expressing the SH3Δ and the N-terΔ mutants had similar numbers of Annexin V+ cells as compared to BCR-ABL inducible cells (32% and 16% v.s.26% with 2 μM IM). Colony forming cell assays (CFC) further showed significantly reduced growth factor independent CFC numbers generated from the SH3WD40Δ mutant compared to BCR-ABL inducible cells co-expressed with the full-length Ahi-1 (4% v.s.46% with 5μM IM compared to untreated cells). Interestingly, a gene expression study comparing full-length Ahi-1-transduced BCR-ABL cells with its mutants further demonstrated that these changes were associated with deregulated expression of Bcl-2 and Cis (Cytokine inducible SH2 protein) involved in apoptosis. These results suggest that the WD40-repeat domain is important for Ahi-1's role in mediating IM-induced apoptosis and that targeting this critical domain of Ahi-1may provide a novel therapeutic option for treatment of CML. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2170 Poster Board II-147 We have previously published that NIH 3T3 cells, which are known to be resistant to Bcr-Abl transformation, are greatly stimulated to undergo Bcr-Abl oncogenic transformation by forced expression of the α and β chains of the human IL-3 receptor (Tao et al., Oncogene 2008). These studies further showed that expression of the receptor chains enhanced activation of Jak2 but had little effect on the ability of Bcr-Abl to activate STAT5, which readily occurred by expression of only Bcr-Abl. Recently we have shown (see Abstract by Samanta et al.) that treatment of Bcr-Abl+ 32D cells with the validated Jak2 inhibitor TG101209 (TargeGen, Inc.) and also with a new Jak2 inhibitor WP1193 for 60-120 min: a) reduced phosphorylation of Tyr 177 of Bcr-Abl and the total level of pTyr Bcr-Abl; b) Bcr-Abl protein levels were rapidly decreased; c) total Ras-GTP, pGab2, pShc and pSTAT5 levels were reduced. These results suggest that major functions attributed to Bcr-Abl are actually controlled by Jak2. However, important questions need to be addressed: a) how is Jak2 regulated in Bcr-Abl+ cells; b) whether activation of Jak2 requires Bcr-Abl only; c) or whether it requires Bcr-Abl associated with IL-3 α and β chains together; and finally d) whether our observations in Bcr-Abl+ mouse hematopoietic cell lines can be reproduced in the Bcr-Abl+, IL-3 receptor+ NIH 3T3 fibroblastic cell line. We observed that NIH 3T3 cells expressing the IL-3 receptor and Bcr-Abl have increased Jak2 activity and increased ability to phosphorylate Tyr 177 of Bcr-Abl compared to NIH 3T3 cells expressing Bcr-Abl only. Phosphorylation of Tyr 177 of Bcr-Abl is needed to activate the Ras and PI-3 kinase pathways. Phosphorylation of Gab2 on Tyr YxxM, which depends on Jak2 kinase activity (Samanta et al., Can Res 2006), plays a critical role in activating the PI-3 kinase pathway. Inhibition of Jak2 with the highly specific Jak2 inhibitor TG101209 and the newly discovered Janus kinase inhibitor WP1193 rapidly reduced phosphorylation of Tyr 177 of Bcr-Abl in Bcr-Abl+ NIH 3T3 cells expressing the IL-3 receptor chains. In Bcr-Abl+ 32D cells Jak2 inhibitors but not imatinib mesylate (IM) inhibited phosphorylation of Bcr-Abl Tyr 177 (see Abstract by Samanta et al.). The lack of IM inhibition of phosphorylation of Tyr 177 supports the conclusion that Bcr-Abl does not autophosphorylate Tyr 177 but Tyr 177 is a target of Jak2. Soft agar clones of NIH 3T3 cells that had expressed both the IL-3 receptor chains and Bcr-Abl in cell culture surprisingly had a strong preference for expressing only the β chain of the IL-3 receptor as agar clones. Five of six clones expressed only the β chain whereas the remaining clone expressed only the α chain. All of these clones expressed Bcr-Abl. The β clones generally had higher levels of activated Jak2 kinase than the α chain clone. Cells expressing the β chain had high levels of pTyr 177 Bcr-Abl. Since the levels of activated Jak2 in NIH 3T3 cells expressing only Bcr-Abl were quite low compared to the cells expressing the IL-3 receptor chains and Bcr-Abl, and since cells expressing β chain only together with Bcr-Abl had higher levels of activated Jak2 compared to the α chain only clone, we propose that the β chain is a critical component utilized by Bcr-Abl to activate Jak2. In this scenario, we hypothesize that the β chain would bind a molecule of Jak2 and Bcr-Abl would bind another molecule of Jak2 (see Xie et al. Oncogene 2001), and furthermore the association of the β chain with Bcr-Abl would facilitate cross phosphorylation of Jak2 at Tyr 1007, resulting in activation of Jak2. Disclosures: No relevant conflicts of interest to declare.