ALBERT

Description: Although an increase in protein phosphorylation on tyrosine was first noted as a result of cell transformation or the application of growth factors to cells, recent reports have shown high levels of tyrosine kinases in nondividing tissues. For that reason, we have investigated whether normal human polymorphonuclear leukocytes (PMN) contain tyrosine kinase and phosphatase activity. Using a copolymer of glutamine: tyrosine as a substrate for the phosphotransferase reaction, we have demonstrated that PMN contain a cytosolic tyrosine kinase activity that elutes as a single peak from Sephacryl S-200 chromatography and has a molecular weight of 70 kilodaltons. Human promyelocytic leukemia cells (HL-60), contain a similar activity (as demonstrated by column chromatography), with only 25% of the activity found in PMN. This cytosolic tyrosine kinase can phosphorylate angiotensin II and a fragment of the src protein containing tyrosine 416, which suggests a similar substrate specificity to other tyrosine- phosphorylating protein kinases. In addition, we have demonstrated that PMN have double the amount of phosphotyrosine phosphatase (PTPase) activity of that found in HL-60 cells. This enzyme has a Km of 0.932 mmol/L and a Vmax of 0.355 mumol inorganic phosphate released/mg protein/min, which is similar to other cellular PTPase. Activation of PMN with f-Met-Leu-Phe and phorbol esters causes a slight but statistically significant drop in PMN PTPase activity. These results suggest that terminally differentiated myeloid cells have high tyrosine kinase and phosphatase activity, which may play a role in stimulus response coupling in the mature PMN.

Description: INTRODUCTIONS: Hematopoietic Stem Cells (HSC) are the most extensively characterized stem cell. Despite our rather extensive knowledge of the HSC, the ability to maintain and expand HSCs in-vitro remains a challenge. The niche, or microenvironment, of the HSC is thought to support and dictate whether HSC will self-renew or differentiate. Many have attempted to create artificial niches, but with limited success. Rather than trying to construct an artificial niche, we sought to examine whether the existent intact bone marrow hematopoietic niche could be maintained and support HSC in-vitro. METHODS: Femurs, and Humeri were removed from healthy 8–12 week old BA Ly5.1. Bone Marrow (BM) preparations were processed as Whole Bone Fragments (WBF), Flushed, or as intact marrow Plugs. Whole bones were cut into thirds to facilitate perfusion. Flushed BM consisted of completely disassociated cells. Lastly, Plugs were flushed intact from the BM cavity. Preparations were cultured in X-Vivo media with 15% FBS, with or without supplemental cytokines (Flt2, TPO, SCF) and media was exchanged every other day. At serial time points, BM preparations were removed and viability determined. Cells analyzed by flow cytometry to determine presence of c-Kit+/Lin−/Sca-1+/(KLS) populations. 1 million cells were removed from preparations and transplanted into sublethally irradiated (400rads) Ly5.2 recipients which after 6 weeks were analyzed for for donor derived engraftment. RESULTS: Under all conditions, viability ranged from 13% to 67% at day 9 in culture, with even higher percentages at day 4 (30–94%). On flow cytometry analysis, populations of KLS cells were detectable in flushed, plug and BMF samples. At day 4 of culture, the frequency of c-Kit+, Sca+ cells of Lin- ranged from .43% to 4.00%. Those cultured with supplemental cytokines showed a significantly higher frequency (.99% to 4.00%) than those without cytokines (.36% to 1.43%). The highest mean frequency of KLS was consistently the Flushed BM at 3.52% Cy- and 4.00% Cy+, suggesting the importance of perfusion. A slight increase in frequency was observed in both Plugs (mean of 1.27%) compared WBF (mean of .67%). There was no negligible difference between Cytokine+ and Cytokine- preparations from WBF at both Day 4 and Day 9 time points. Day 9 frequency of KLS cells fell to the range of .29% to 1.23%. However, frequencies decreased proportionally amongst all samples. CFU assay scoring demonstrated the presence of colonies from both Day 4 and 9 samples. In sublethally irradiated mice transplanted with cultured marrow cells, tri-lineage donor derived Ly5.1 cells were detectable at up to3 months post transplant from both flushed marrow plugs and WBF from D+9 culture preparations. Donor granulocyte engraftment from WBF donor cells (without cytokines) was 5.2% to 22.1, as compared to 19% from an equal number of fresh control BM cells, suggesting long-term engraftment was derived from HSC maintained in culture WBF. CONCLUSIONS: These results suggest that the hematopoietic niche can be maintained in whole bone fragments up to 9 days in culture with minimal manipulation and without cytokine supplementation. Viability however decreases over time, suggesting that in order to maintain a more robust niche, means to increase intact niche perfusion will be necessary.

Description: There were 26 patients enrolled in a pilot study of high-dose immunosuppressive therapy (HDIT) for severe multiple sclerosis (MS). Median baseline expanded disability status scale (EDSS) was 7.0 (range, 5.0-8.0). HDIT consisted of total body irradiation, cyclophosphamide, and antithymocyte globulin (ATG) and was followed by transplantation of autologous, granulocyte colony-stimulating factor (G-CSF)-mobilized CD34-selected stem cells. Regimen-related toxicities were mild. Because of bladder dysfunction, there were 8 infectious events of the lower urinary tract. One patient died from Epstein-Barr virus (EBV)-related posttransplantation lymphoproliferative disorder (PTLD) associated with a change from horse-derived to rabbit-derived ATG in the HDIT regimen. An engraftment syndrome characterized by noninfectious fever with or without rash developed in 13 of the first 18 patients and was associated in some cases with transient worsening of neurologic symptoms. There were 2 significant adverse neurologic events that occurred, including a flare of MS during mobilization and an episode of irreversible neurologic deterioration after HDIT associated with fever. With a median follow-up of 24 (range, 3-36) months, the Kaplan-Meier estimate of progression (≥ 1.0 point EDSS) at 3 years was 27%. Of 12 patients who had oligoclonal bands in the cerebrospinal fluid at baseline, 9 had persistence after HDIT. After HDIT, 4 patients developed new enhancing lesions on magnetic resonance imaging of the brain. The estimate of survival at 3 years was 91%. Important clinical issues in the use of HDIT and stem cell transplantation for MS were identified; however, modifications of the initial approaches appear to reduce treatment risks. This was a heterogeneous high-risk group, and a phase 3 study is planned to fully assess efficacy. (Blood. 2003;102:2364-2372)

Description: Abstract 2445 Constitutive activation of signal transducer and activator of transcription 3 (STAT3) correlates with drug resistance and a poor prognosis in many cancers. STAT3 signaling is mediated by phosphorylation at tyrosine-705 (p STAT3Y705), dimerization, and nuclear transactivation. In chronic myeloid leukemia (CML), pSTAT3Y705 is demonstrable under two distinct resistance scenarios: (1) extrinsic resistance, in which BCR-ABL1 kinase-independent survival signals originating from the bone marrow (BM) microenvironment activate pSTAT3Y705 in a JAK2- or TYK2-dependent manner, and (2) intrinsic resistance, in which BCR-ABL1 kinase-independent signals activate pSTAT3Y705 in response to kinase inhibition. Based on these observations, we identified TKI-resistant CML as an excellent model for developing and optimizing pharmacologic STAT3 inhibitors. Using K562 and AR230 CML cells that are resistant to 1 μM imatinib (K562R and AR230R; intrinsic resistance) and primary CML CD34+ progenitor cells exposed to BM stromal-derived conditioned medium (CM; extrinsic resistance), we examined the effects of direct pharmacologic inhibition of STAT3 in TKI-resistant CML. Here, we report the design and validation of next-generation STAT3 inhibitors identified through computational modeling and screening in AR230R CML cells expressing high levels of pSTAT3Y705. We initially examined the effects of an established STAT3 inhibitor, S3I-201.1066 (SF1–066). K562R or AR230R cells were treated with 1 μM imatinib and/or 10 μM SF1–066, followed by culture in methylcellulose medium and scoring after 14–16 days. Combination treatment reduced the clonogenicity of K562R and AR230R cells to 31.4% (p

Description: Abstract 2303 Background: The genes and pathways that govern the functions and expansion of hematopoietic stem cells (HSC) remain to be elucidated. Pim (proviral insertion in murine lymphomas) protein kinases are a small family of constitutively active, highly conserved oncogenic serine/threonine kinases and have 3 members: Pim1, Pim2, and Pim3. Currently, little is known about the contribution of each Pim kinase in hematopoiesis. Pim1 was recently found to be important in the regulation of CXCR4 expression in HSCs (Grundler R et al, J. Exp Med 2009, 206:1957). However, the roles of Pim1 in HSC proliferation, self-renewal, and long-term repopulation are unclear. In the current study, we performed detailed hematological and bone marrow transplant studies to address these questions. Methods: We generated Pim1 transgenic (Tx) mice bearing human PIM1 under the control of vav hematopoietic promoter (vav-hPIM1 Tx). Pim1−/−, Pim2−/−, Pim3−/− single knockout (KO) mice were also utilized. We quantified the number of hematopoietic stem/progenitor cells (HSPCs) in these mice using flow cytometry, colony forming units (CFUs) and cobblestone area forming cell colonies (CAFCs) assays. In vivo BrdU labeling was performed to determine the proliferation status of long-term HSCs in these mice. CXCR4 expression and the homing of HSCs to bone marrow and spleen were also analyzed. Additionally, we performed noncompetitive, competitive, and serial transplantation assays to examine the role of Pim1, Pim2 and Pim3 in hematological reconstitution in lethally irradiated recipient mice. Finally, a limiting dilution competitive bone marrow transplantation assay using purified long-term HSC cells (Lin− Sca-1+c-kit+ CD34−) was performed to calculate the frequency of active HSCs in vav-hPIM1 Tx and Pim1−/−mice. Results: Conclusion: Our current studies demonstrate a novel role of Pim1 serine/threonine kinase in the regulation of self-renewal, proliferation, and long-term repopulation of HSCs. The functions of Pim1 in hematopoiesis do not overlap with Pim2 or Pim3. Acknowledgment: We thank Richard Peppler at the Hollings Cancer Center Flow Cytometry Core for performing flow cytometry analysis. This work is supported by MUSC Hollings Cancer Center Startup Fund, Hollings Cancer Center ACS IRG, ASCO Conquer Cancer Foundation Career Development Award, NIH 1K08HL 103780–01A1, and NIH 3P30CA138313–01S3. Disclosures: No relevant conflicts of interest to declare.

Description: Allogeneic hematopoietic cell transplantation (HCT) can cure a variety of benign and malignant hematopoietic disorders, but graft-versus-host disease (GVHD) remains a significant source of transplant-related mortality and morbidity. Mature T cells in donor stem cell graft are primarily responsible for the development of acute GVHD. PIM kinases are a family of serine/threonine kinases, including PIM1, PIM2 and PIM3, which are expressed following T-cell activation. The PIM kinases have been shown to inhibit apoptosis as well as to stimulate the cell metabolism and protein synthesis. In the current study, we investigated how PIM kinases regulate T-cell responses to alloantigens and GVHD development. Using genetically modified mice on an FVB background that are deficient for either single, double, or triple kinases of the PIM family, we evaluated the role of PIM kinases on T-cell alloresponses. T cells deficient for PIM1/PIM3 kinases had reduced survival and proliferation upon stimulation with alloantigens in vitro, whereas T cells without the PIM2 kinase displayed increased survival and IFNγ production as compared to WT T cells. After being transferred to lethally irradiated allogeneic mice in vivo, PIM1/PIM3 double knockout (KO) T cells had reduced expression of IFNγ in spleen, whereas PIM2 KO T cells showed decreased IL-4/5 expression, although similar IFNγ production when compared with WT T cells. These data suggest PIM2 kinase plays a distinct role by negatively regulating T-cell alloresponses. To test the role of PIM kinases on T-cell ability to induce GVHD, we transferred WT, PIM2 single KO, PIM1/PIM2 double KO, PIM1/PIM3 double KO, PIM2/PIM3 double KO, or PIM1/PIM2/PIM3 triple KO T cells together with WT bone marrow cells into lethally irradiated B6 mice. While PIM1/PIM3 double KO T cells induced milder GVHD, PIM2 KO T cells induced much more severe GVHD when compared to WT T cells. In addition, any types of T cells deficient for PIM2 caused significantly more severe GVHD than their PIM2-replete counterparts. Furthermore, restoration of PIM2 expression by gene transfection reduced the ability of PIM2-deficient T cells in the induction of GVHD. Enhanced pathogenicity of PIM2-deficient T cells was also confirmed in another murine model of allogeneic HCT. These results indicate that PIM2 plays a dominant role among PIM kinases in negatively regulating T-cell alloresponses and GVHD induction. To validate the data obtained from KO mice, we silenced the PIM2 kinase in PIM1/PIM3 KO T cells by administration of a PIM kinase pan-inhibitor (AZD, AstraZeneca R&D, Waltham, MA), and we observed exacerbated GVHD induced by PIM1/PIM3 KO T cells after PIM2 kinase silencing. Our study demonstrates the important result that PIM2 negatively regulates T-cell mediated alloresponse and GVHD development. The finding uncovers a novel biological function of PIM kinases as well as urges caution in inhibiting PIM kinases for the treatment of hematologic malignances in patients after allogeneic HCT. YW and AD contributed equally to this work. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 293 The serine/threonine Pim kinases are known to play an important role in several signal transduction pathways, including those regulated by c-Myc, N-Myc, FLT3-ITD, BCR-ABL, HOXA9, and EWS fusions. Pim kinases are up regulated in some hematologic malignancies such as Acute Myeloid Leukemia (AML) and Chronic Lymphocytic Leukemia. Pim kinases were first identified as a proviral integration site in the mice that overexpress c-Myc and enhance lymphomagenesis. We have previously shown that SMI-4a, a novel benzylidene-thiazolidine-2,4-dione small molecule inhibitor of the Pim kinases inhibited the growth of precursor T-cell lymphoblastic leukemia in the mouse (Lin, YW. et al. Blood, 2010). We have also reported that SMI-4a in combination with a few MEK inhibitors or standard chemotherapeutic agents including daunorubicin, Ara-C, and 6-thioguanine synergistically killed myeloid and lymphoblastic leukemic cell lines as well as primary patient leukemic blasts (Lin, YW. et al. Blood, 2010, AACR 2010 abstract). In the current study, we found that the combination of Rapamycin and Pim kinase inhibitors including SMI-4a, SMI-20a, SMI-24a, and K00135 synergistically killed MV4-11, a myeloid leukemia cell line harboring FLT3-ITD. We also show that a combination of SMI-4a and Rapamycin significantly inhibited subcutaneous tumor growth of MV4-11 expressing firefly-luciferase in NOD/SCID mice, which was determined using conventional caliper measurement and bioluminescent analysis in the region of interest. Although the combination of those drugs caused a loss of appetite in NOD/SCID mice in the first week of the treatment, the mice became tolerable and did not lose weight from the second week. In addition, none of those drugs alone or in combination caused any adverse affects in wild type FVB mice. A combination of SMI-4a and Rapamycin also synergistically kills primary AML blasts including patients with or without expression of the FLT3-ITD. The treatment with SMI-4a or Rapamycin as a single agent down regulates phosphorylation of two substrates of the mTORC1 pathway, 4E-BP1 and S6K, while the combination significantly decreased their phosphorylation compared with single agent treatments, which is consistent with the synergistic effect. We found that SMI-4a increased phosphorylation of AMPK and decreased phosphorylation of mTOR at serine 2448 leading to down regulation of the mTORC1 pathway. In contrast, Rapamycin induced down regulation of 4E-BP1 and S6K without affecting phosphorylation of AMPK and mTOR. SMI-4a induced apoptotic cell death that was characterized by down regulation of MCL1, cleavage of Caspase 3, and nuclear condensation, whereas Rapamycin did not induce these changes. Together those two inhibitors modify different signaling pathways to synergistically kill AML blasts in vitro and in vivo. Disclosures: Tholanikunnel: Vortex Biotechnology: Employment. Kraft:Vortex Biotechnology: Consultancy.

Description: A β-catenin expression signature is associated with primary resistance to tyrosine kinase inhibitors (TKIs) (McWeeney et al. 2010), but its role in TKI resistance is not completely understood. To assess the role of β-catenin in TKI resistance, we used shRNA targeting β-catenin (shβcat) in in vitro models of BCR-ABL1 kinase-independent resistance. To model resistance in the absence of bone marrow (BM)-derived factors, we used TKI-resistant K562R cells that are adapted for growth in the presence of imatinib, as well as primary CD34+ progenitors from CML patients who had failed treatment with two or more TKIs. These cells are cultured in regular medium (RM), proliferate in 1.0-2.5 μM imatinib, and exhibit β-catenin expression that is independent of BCR-ABL1 kinase activity. To model resistance mediated by the BM microenvironment, we cultured TKI-sensitive parental K562 cells and CD34+ progenitors from newly diagnosed CML patients in direct contact (DC) with HS-5 BM stromal cells. HS-5 co-culture increases β-catenin protein levels and clonogenic potential by 〉3-fold despite continued suppression of BCR-ABL1 kinase activity. All cells lack BCR-ABL1 kinase domain mutations and undergo TKI-mediated kinase inhibition as detected by immunoblot analyses. CML cell lines and primary cells with BCR-ABL1-independent resistance were lentivirally transduced with shβcat or a scrambled control (shSCR), and knockdown was confirmed by immunoblot and/or qRT-PCR. In RM, shβcat reduced the clonogenicity of TKI-sensitive K562 cells and CD34+ cells from newly diagnosed CML patients by 49% and 39%, respectively, compared to shSCR controls. In TKI-resistant K562R cells and CD34+ cells from TKI-resistant CML patients, shβcat reduced clonogenicity by 60% and 50%, respectively, in the presence or absence of imatinib (0-10 μM), suggesting a role for β-catenin in the development or maintenance of TKI resistance. In contrast to cells grown in RM, clonogenicity of cell lines and patient samples cultured in HS-5 DC was unaffected by shβcat compared to imatinib alone. Immunoblot analyses revealed that β-catenin protein levels were fully restored in HS-5 DC, despite the continued presence of shβcat. qRT-PCR revealed that while cells in HS-5 DC have high amounts of β-catenin protein, the mRNA levels remained similar to shβcat-expressing cells cultured in RM, consistent with post-translational stabilization of β-catenin. Importantly, increased β-catenin was not observed when cells were cultured in HS-5 conditioned medium, indicating that stabilization requires DC with the bone marrow stroma. These data are consistent with a role for β-catenin in TKI resistance mediated by DC with the BM microenvironment, similar to a recent report (Zhang et al., 2013). To understand nuclear versus cytoplasmic distribution following HS-5 DC, CD34+ cells from newly diagnosed CML patients were cultured in RM or HS-5 DC for 36 hours and analyzed for β-catenin localization by immunfluorescence. As expected, cells cultured in RM had low levels of β-catenin in the nucleus and cytoplasm that decreased upon treatment with imatinib (2.5 μM). In contrast, cells cultured in HS-5 DC had a marked increase of β-catenin that was unaffected by treatment with imatinib. While detectable in the nucleus, the majority of β-catenin protein was localized in the cytoplasm and at the cell membrane, consistent with its role in cell-cell junctions. Accordingly, in CD34+ cells from newly diagnosed CML patients, an N-cadherin blocking antibody impaired the clonogenic potential of cells cultured in HS-5 DC, with no significant effect on cells grown in RM. Affymetrix Human Gene 1.0 ST arrays revealed high levels of genes encoding the CDH2 and CDH13 cadherins, which may be involved in N-cadherin-mediated β-catenin stabilization, even in the presence of shβcat. Preliminary data also suggests that HS-5 DC reduces luciferase reporter activity from a construct harboring sequential β-catenin binding elements (pGF1-Lef/Tcf-eFGP-luc), further supporting a role for cytoplasmic β-catenin in TKI resistance. These data demonstrate a critical role for β-catenin in BCR-ABL1 kinase-independent TKI resistance, and suggest new strategies for targeting TKI resistance in the absence of BCR-ABL1 kinase domain mutations. Disclosures: Deininger: Bristol-Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding.

Description: New approaches are needed to treat and cure T-cell acute lymphoblastic leukemia (T-ALL) patients who are currently failing to achieve a remission with chemotherapy. While very intensive chemotherapy regimens result in remission in many T-ALL patients, early T-cell precursor (ETP)-ALL, defined by the absence of CD4, CD8, and CD1a and frequent expression of one or more myeloid markers, is associated with a higher rate of relapse and induction failure with a 10-year overall survival of 19% as compared with 84% for all other T-ALLs. The PIM serine/threonine protein kinase has been identified as a potential novel target for the development of new treatment regimens for the cure of hematopoietic malignancies. To explore the potential association of elevated PIM1 expression with ETP-ALL, we analyzed a publically available St. Judes gene expression dataset GSE28703. Results demonstrated that PIM1 is highly expressed in 75% of patients with ETP-ALL and in 13% of non-ETP ALL. PIM1 overexpression in immature or ETP-ALL is further validated using two other publically available independent cohorts: E-MEXP-313 and GSE62156. Based on this observation, we then tested the ability of pan-PIM inhibitors (AZD1208/LGB-321) on the growth of 6 human T-ALL cell lines. qRT-PCR and western blot analysis confirmed, PIM inhibitor sensitive (XTT assay: IC50 〈 1 micromolar) cell lines (H-SB2, DU.528, and KOPT-K1) had elevated levels of PIM1 (〉9 folds, p 10 micromolar) cell lines express significantly low PIM1, activated NOTCH mutations, activated AKT signaling, and higher levels of MYC. Immunophenotype of all the PIM inhibitor insensitive cells lines was consistent with a more mature T-ALL phenotype (CD4+/CD8+ and TdT+). When we selected NOTCH-mutant mature T-ALL cells for resistance to gamma secretase inhibitors (SUP-T1-Persister cells) they developed elevated PIM1 kinase levels and were sensitive to the growth inhibitory activities of PIM inhibition. Thus, PIM appears to be a potential target for chemotherapeutic treatment intervention in T-ALL and especially ETP-ALL tumor cells. While PIM kinase inhibitors block ETP-ALL growth they do not induce cell death. The observation that treatment with PIM inhibitors activated ERK and STAT phosphorylation, along with the knowledge that T-ALL cells have increased levels of SRC family tyrosine kinases suggested that adding a broadly active tyrosine kinase inhibitor could enhance the killing of these tumor cells. We have made the novel observation that the combination of Ponatinib, a tyrosine kinase inhibitor used in the treatment of chronic myelogenous leukemia, with a PIM kinase inhibitor is synergistically lethal to ETP-ALL cells (CI 〈 1.0). These agents synergize to induce PARP and Caspase cleavage, sufficient to drive apoptosis. In murine models, following engraftment of NOD/scid/IL-2Rɣ null (NSG) mice with H-SB2-luc cell xenografts, three weeks of dual therapy with AZD1208 (30mg/kg/day) and Ponatinib (3mg/kg/day) significantly blocked leukemia growth as evidenced by optical scanning for luciferase producing cells (Figure 1, p

Description: Abstract 31 In CML, TKI resistance in the absence of BCR-ABL1 kinase mutations is mechanistically unclear. Since extrinsic signals from the bone marrow (BM) microenvironment protect CML cells from TKIs in a STAT3-dependent manner (Bewry et al. 2008; Traer et al. 2011), we hypothesized that overt resistance may occur when CML cells maintain intrinsic STAT3 activation in the absence of extrinsic signals. We also asked whether combined targeting of STAT3 and BCR-ABL1 could produce synthetic lethality to overcome TKI resistance. To model extrinsic BCR-ABL1 kinase-independent resistance, we grew CML cell lines and CMLCD34+ progenitor cells from newly diagnosed patients in the presence of conditioned medium (CM) derived from HS-5 BM stromal cells. To model intrinsic resistance, we used the imatinib-resistant K562R and AR230R cell lines. Our models of TKI resistance proliferate in 1.0–2.5 μM imatinib and exhibit pSTAT3Y705 activation despite suppression of BCR-ABL1 kinase activity. To investigate the role of pSTAT3Y705 in TKI resistance, we used shRNA-mediated knockdown (shSTAT3), a dominant-negative mutant (dnSTAT3), and pharmacologic STAT3 inhibitors developed in our laboratories to block STAT3 activity. Compared to scrambled controls in the presence of HS-5 CM, shSTAT3 reduced colony formation (33.8% reduction, p