ALBERT

Description: CLL remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Honokiol is a natural product known to possess potent anti-neoplastic and anti-angiogenic properties. We examined whether Honokiol can overcome apoptotic resistance in CLL cells. Honokiol induces caspase-dependent cell death of CLL cells, characterized by an increase in Annexin V positive, propidium iodide negative cells that was blocked by the caspase inhibitor, Z-VAD-fmk. Further evidence for Honokiol-induced apoptosis of CLL cells was confirmed by an increase in caspase 3 activity and cleavage of PARP. In addition, Honokiol demonstrated an LC50 that was lower for CLL cells than for normal mononuclear cells, suggesting that CLL cells are more susceptible to the cytotoxic effects of Honokiol compared to normal hematopoietic cells. To determine whether Honokiol-induced apoptosis of CLL cells was associated with known positive therapeutic effects, we examined mcl-1 expression, a survival protein whose down-regulation is associated with response to treatment in CLL patients. Honokiol treatment of CLL cells resulted in a significant decrease in the expression of mcl-1 within 24 hours. Furthermore, CLL cells pre-treated with IL-4, a cytokine known to support CLL survival, underwent apoptosis when subsequently incubated with Honokiol, indicating that Honokiol could also overcome the pro-survival effects of IL-4. These data indicate that Honokiol is a potent inducer of apoptosis in CLL cells and should be examined for further clinical application.

Description: Bryostatin 1 is known to exhibit in vitro and in vivo activity against chronic lymphocytic leukemia (CLL) cells by inducing their further maturation into plasmalike cells. Signal transducer and activator of transcription (STAT) proteins play a central role in B-lymphocyte growth and function and are aberrantly phosphorylated on serine residues in CLL cells. To determine whether STAT transcription factors are important in Bryostatin 1–induced differentiation of CLL cells, primary CLL cells were examined for signaling events following exposure to Bryostatin 1 in vitro. Western analysis and electrophoretic mobility shift assays revealed that Bryostatin 1 induced tyrosine phosphorylation and DNA binding of STAT1, yet there was no effect on constitutive serine phosphorylation of STAT1. Bryostatin 1–induced STAT1 activation occurred in a manner that was dependent on protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Janus tyrosine kinase (JAK) activation. Evidence indicates that Bryostatin 1 induces STAT1 activation through an interferon γ (IFNγ) autocrine loop. However, STAT1 activation by IFNγ stimulation alone was not sufficient to induce differentiation. This insufficiency is due to the broader effect on gene expression caused by Bryostatin 1 compared with IFNγ, as demonstrated by microarray analysis. Both up-regulation of CD22 expression and immunoglobulin M (IgM) production, markers of CLL differentiation, were inhibited by a decoy oligonucleotide for STAT1, indicating that STAT1 is necessary for Bryostatin 1–induced differentiation of CLL cells. This study implicates STAT transcription factors as important mediators of Bryostatin 1–induced differentiation of CLL cells and could possibly lead to improved therapeutic approaches for the treatment of CLL.

Description: Although much progress has been made in recent years in understanding the molecular pathogenesis of CLL, new therapeutic approaches are critical to improving patient outcome. One attractive approach is differentiation therapy, in which the malignant B cells are induced to mature into non-replicative cells with a shorter lifespan. We have shown previously that the natural product Bryostatin 1 induces differentiation of CLL cells through activation of the transcription factor STAT1. To identify genes activated by STAT1 that mediate Bryostatin 1-induced differentiation of CLL cells we used gene expression microarrays. Using this approach, we identified seven genes that were potential STAT1 target genes, including GADD45β, IFI16, caspase 3, topoisomerase I, and the known STAT1 target IRF-1. Given the limitations of genetically manipulating primary CLL cells, we evaluated whether B lymphocytic cell lines recapitulated the molecular and cellular differentiation responses to Bryostatin 1 that we observed in CLL cells. We found that Bryostatin 1 is a potent inducer of differentiation of CESS B lymphoblastoid cells, as measured by growth arrest, downregulation of MHC II, and IgG secretion. Bryostatin 1 also induced signaling events in CESS cells similar to those observed in CLL cells, including activation of STAT1 and induction of STAT1 target genes. These findings suggest that STAT1 and its targets may be important mediators of malignant B cell differentiation and that the CESS cell line can be used to further study the potential clinical application of differentiation therapy in CLL.

Description: Abstract 1905 Poster Board I-928 Myeloproliferative neoplasms (MPN) are a group of clonal disorders that arise from the transformation of hematopoietic stem cells. The majority of patients with MPN show a mutation in the Jak2 tyrosine kinase (Jak2V617F), which results in the constitutive activity of this kinase. This mutation is believed to play a critical role in the pathogenesis of these disorders, and therefore, the development of Jak2 kinase inhibitors has been a high priority. Jak2 directly phosphorylates the transcription factor STAT5, and it is hypothesized that STAT5 activation is required for Jak2V617F mediated transformation. Since STAT5 is a critical mediator of the effects of Jak2V617F, the development of drugs that inhibit this transcription factor holds promise as a treatment for MPN, and the dual inhibition of both STAT5 and Jak2 may yield better results with less toxicity. We previously identified the neuroleptic drug pimozide as an inhibitor of STAT5 transcriptional function in a cell based screen. In order to determine the potential of pimozide as a STAT5 inhibitor in MPN cell models, we utilized Ba/F3 cells reconstituted with the Jak2V617F mutation (Ba/F3EJ) as well a human erythroleukemia cell line (HEL) harboring the Jak2V617F mutation. Ba/F3EJ and HEL cells showed a dose dependent decrease in STAT5 tyrosine phosphorylation when treated with pimozide. In addition, pimozide decreased the expression of key STAT5 target genes, such as Bcl-xl, Mcl1, CyclinD1 and Pim1. Moreover, pimozide induced a dose dependent reduction in cell viability in both cell lines. Pimozide induced both G0/G1 arrest as well as apoptosis as manifested by increased caspase activity and increased annexin V/PI staining. We hypothesized that dual inhibition of both Jak2 and STAT5 may lead to enhanced cytotoxic effects on myeloproliferative cells. Indeed, combination treatment with pimozide and Jak inhibitor 1 led to a greater inhibition of the tyrosine phosphorylation of STAT5, and a bigger reduction in the level of the STAT5 target protein Mcl1. This dual inhibition of the Jak-STAT pathway led to enhanced toxicity to the myeloproliferative cells. 10 uM pimozide led to a 30% reduction in the number of viable HEL cells at 48 hours, and 0.8 uM JAK inhibitor 1 led to a 37% reduction in viable cell number. Significantly, the combination of both drugs led to an 83% reduction in viable cells. Furthermore, this combination led to an increase in apoptosis as measured by caspase cleavage and flow cytometric analysis of annexin V staining. The number of annexin V positive cells treated for 48 hours with the combination of pimozide and Jak Inhibitor 1 was greater than 3 times compared to each drug alone in Ba/F3EJ cells and was increased 2.5 fold in HEL cells. In conclusion, pimozide inhibits STAT5 activation in MPN cells and effectively reduces the number of viable cells by inducing apoptosis. These effects are enhanced when pimozide is combined with Jak2 inhibition. These data suggest that directly inhibiting STAT5, as well as the combination of inhibiting both STAT5 and Jak2, may be effective strategies for the treatment of MPN. Disclosures: Off Label Use: We describe in vitro data showing that the neuroleptic drug pimozide shows anti-tumor activity on MPN cells..

Description: Activation of the transcription factor STAT3 is essential for the pathogenesis of many cancers, including multiple myeloma. While normal cells can tolerate a reduction in STAT3 function, tumors often require constitutive STAT3 signaling for survival. Thus, identifying drugs that inhibit STAT3 activity may provide new therapeutic agents useful for cancer treatment. We have developed a high throughput cell-based screen to identify drugs that inhibit STAT3-dependent transcriptional activity. To assure the specificity of these drugs for STAT3 function, we performed a counter screen assessing NF-kappaB-dependent transcriptional activity. To bypass the difficulties inherent in the development of novel small molecules for clinical use, we analyzed a library of 1120 drugs that are either FDA approved, or are otherwise known to be safe in humans. From this screen, we identified nifuroxazide, a drug used to treat dehydration associated with diarrheal illness, as a potent inhibitor of STAT3 transcriptional activity. By contrast, nifuroxazide has no effect on NF-kappaB-dependent transcription. Myeloma cells containing constitutive STAT3 activation show decreased STAT3 tyrosine phosphorylation when incubated with 10 uM nifuroxazide. In addition, expression of STAT3 target genes necessary for myeloma survival, including bcl-x, mcl-1, and cyclin D1, is markedly reduced by 10 uM nifuroxazide. To determine whether these effects of nifuroxazide on STAT3 signaling alter cell viability, we utilized U266 myeloma cells, which depend on STAT3 activation for survival. U266 viability is inhibited by nifuroxazide at an EC50 of approximately 3 uM. Notably, RPMI 8226 myeloma cells, which do not contain activated STAT3, are not affected by comparable concentrations of nifuroxazide. In addition, this dose has no effect on normal peripheral blood mononuclear cells. Given that myeloma cells receive survival signals from bone marrow stromal cells, we determined if nifuroxazide affects myeloma survival in stromal cell co-cultures. Nifuroxazide is effective at reducing U266 viability in the presence of bone marrow stromal cells at an EC50 of approximately 3 uM. Thus, screening for compounds that inhibit STAT3 transcriptional activity is useful in identifying potential drugs for myeloma therapy. Through this approach, we have identified a novel STAT3 inhibitory function for nifuroxazide. Nifuroxazide inhibits STAT3 mediated survival of myeloma cells and may be useful, either alone or in combination with other drugs, for the treatment of patients with multiple myeloma.

Description: B-cell chronic lymphocytic leukemia (B-CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Honokiol is a natural product known to possess potent antineoplastic and antiangiogenic properties. We examined whether honokiol can overcome apoptotic resistance in primary tumor cells derived from B-CLL patients. Honokiol induced caspase-dependent cell death in all of the B-CLL cells examined and was more toxic toward B-CLL cells than to normal mononuclear cells, suggesting greater susceptibility of the malignant cells. Honokiol-induced apoptosis was characterized by the activation of caspase-3, -8, and -9 and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP). Exposure of B-CLL cells to honokiol resulted in up-regulation of Bcl2-associated protein (Bax) and down-regulation of the expression of the key survival protein myeloid-cell leukemia sequence 1 (Mcl-1), which is associated with response to treatment in B-CLL patients. In addition, B-CLL cells pretreated with interleukin-4 (IL-4), a cytokine known to support B-CLL survival, underwent apoptosis when subsequently incubated with honokiol, indicating that honokiol could also overcome the prosurvival effects of IL-4. Furthermore, honokiol enhanced cytotoxicity induced by fludarabine, cladribine, or chlorambucil. These data indicate that honokiol is a potent inducer of apoptosis in B-CLL cells and should be examined for further clinical application either as a single agent or in combination with other anticancer agents. (Blood. 2005;106:690-697)

Description: Despite advances in targeted therapy, chronic lymphocytic leukemia (CLL) remains a highly prevalent and essentially incurable malignancy. One molecular hallmark of CLL is the constitutive serine phosphorylation and transcriptional activation of the oncogenic transcription factor STAT3. Targeting STAT3 may have a high therapeutic index since normal cells can tolerate a loss of STAT3 function. To identify STAT3 inhibitors that could be rapidly introduced into proof-of-concept clinical trials, we screened a chemical library of drugs known to be safe in humans for specific inhibitors of STAT3-dependent transcription. Using this strategy, we identified the anti-parasitic agent pyrimethamine as a drug that could inhibit STAT3 at levels safely achieved for months at a time in humans. Pyrimethamine inhibited STAT3-dependent gene expression in CLL cells ex vivo, and decreased survival of CLL cells, but not peripheral blood mononuclear cells from healthy donors. To determine whether STAT3 inhibition would confer clinical benefit in patients with CLL, we conducted a phase I clinical trial of continuous daily pyrimethamine, in the era before targeted therapy, in relapsed CLL patients whose disease progressed despite standard therapies. We used a typical 3+3 dose escalation design with three cohorts, 12.5, 25 and 50 mg per day (mg/d). Samples for PK/PD analysis were drawn weekly in the first month and every other week in the second. Sixteen heavily pretreated patients enrolled on the phase 1 portion of this study. The median age was 63 (36-85) and the median time from diagnosis to study therapy was 74 months (range, 6-176 months). Six patients had 17p deletion and 4 had 11q deletion, while 12 of 13 evaluable patients had unmutated IGHV. The patients had a median of 6 prior therapies, including 8 patients who had received prior high dose methylprednisolone, five who had received prior alemtuzumab, and one each with prior allogeneic and autologous stem cell transplantation. Three patients each enrolled on cohorts 1 and 2, and ten patients enrolled on cohort 3. There were no dose limiting toxicities and no significant drug-related toxicities. No objective responses by IW-CLL 2008 criteria were observed. Half of patients achieved stable disease, with one patient dosed at 50 mg/d on therapy for 12 months, and two at 25 mg/d on therapy for 4 and 6 months. The remaining patients had progressive disease, and all but one patient discontinued therapy for progressive disease. The median time on therapy was 1.1 months (0.23-9.99), with median progression free survival of 1.5 months (0.92-5.52) and median overall survival of 22 months (11.75-NA). To determine whether the levels of pyrimethamine achieved in patients corresponded with levels known to inhibit STAT3 transcriptional function in vitro, we measured trough concentrations of pyrimethamine in plasma and in peripheral blood mononuclear cells at distinct time points for patients on the trial. Plasma concentrations and intracellular concentrations were highly correlated, and also correlated closely with dose level. Only in the highest dose cohort did the plasma concentrations reproducibly approach the threshold of 10 micromolar at which STAT3 inhibition occurred consistently in vitro. To determine whether pyrimethamine was exerting an on-target effect in inhibiting STAT3-dependent gene expression in vivo, we first defined a signature of five STAT3-dependent genes (AIM2, ATXN1, ENPP2, GAB1, and ID3) that are upregulated in CLL cells relative to normal B lymphocytes. We then determined expression of these genes from the patients' CLL cells at baseline and while on pyrimethamine. In addition, to further generate predictors of response, we also quantitated the cytotoxic effect of pyrimethamine on the pre-treatment cells of patients ex vivo. We then correlated these potential predictive and pharmacodynamic biomarkers with drug levels and clinical course. In conclusion, pyrimethamine is an inhibitor of STAT3 transcriptional function that can safely be given to patients with CLL. It may be necessary to increase the daily dose of pyrimethamine to beyond 50 mg/day to adequately determine whether this drug can exert an on-target therapeutic effect in CLL patients. Since STAT3 target genes include many immune-suppressive and anti-apoptotic genes, it is likely that pyrimethamine will be most beneficial when combined with other therapeutic modalities. Disclosures Brown: Pharmacyclics: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Boehringer: Consultancy; Sun Pharmaceutical Industries: Research Funding; Gilead: Consultancy, Research Funding; Loxo: Consultancy; Verastem: Consultancy, Research Funding; Invectys: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy; Janssen: Consultancy; Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Sunesis: Consultancy; Genentech: Consultancy; Beigene: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Consultancy. Wu:Neon Therapeutics: Equity Ownership.

Description: Alternative pre-mRNA splicing (AS) is a normal epigenetic phenomenon, a key regulator of gene expression, yields multiple transcripts and thus a variety of proteins from a single gene. Mutations in the spliceosome components resulting in aberrant splicing isoforms are common in AML, and other myeloid neoplasms, and may generate leukemia-specific neoantigens targetable with an antibody-drug conjugates (ADCs) or blocking antibodies. Our previous studies revealed that the FLT3 cell surface receptor is one of the most commonly misspliced genes in AML (54-63% of ~400 AML patients). We conducted cloning and sequencing analyses in AML cells and identified multiple aberrant splice-variants of FLT3 that resulted from either skipping of one or more exons or activation of cryptic splicing sites. Transfection of cDNA with three of these variants in TF-1 (AML cell line) cells resulted in expression of Flt3 variant proteins on the cell surface. We successfully generated rabbit polyclonal antiserum against a unique peptide sequence present in the most commonly expressed abnormal splice variant, which we termed Flt3Va. Immunoblots performed with the polyclonal antibody identified a ~160 kDa protein expressed by TF-1 cells transfected with FLT3Va, and the antibody did not react with untransfected TF-1 cell lysate. Using standard techniques, we generated rabbit hybridomas and evaluated the clones by flow cytometry and western blotting experiments. Based on these data, we selected one antibody clone (15-7) for further experiments. The 15-7 anti-Flt3Va rabbit monoclonal antibody identified Flt3Va protein expressed on the cell surface and within the cytoplasm of transfected TF-1 cells by flow cytometry and western blotting. However, no Flt3Va protein was detected in untransfected TF-1 cells or normal CD34+ bone marrow cells. The 15-7 antibody bound to 26 of 52 primary AML samples and 5 of 10 primagraft samples (PDX models) of human AML. Immunoblotting analyses of PDX models and patient samples confirmed binding to a protein of the expected size (130-160 kDa). Additionally, multi-parameter flow cytometry in 10 PDX models and 52 primary demonstrated that putative AML stem cells (as defined by the CD45dim, CD34, CD38, CD33, c-Kit cell surface expression) co-expressed Flt3Va antigen in 50% samples evaluated. An analysis of Flt3Va protein localization by live cell imaging showed a punctate distribution of Flt3Va on the cell surface. Furthermore, we observed that overexpression of Flt3Va in TF-1 cells led to GM-CSF growth factor independence. Analysis of TF-1 cells in the absence of GM-CSF and Flt3 ligand demonstrated constitutive activation of STAT5, an important mediator of Flt3 signaling, in Flt3Va overexpressing cells. In addition, Erk1/2 phosphorylation was also increased in Flt3Va overexpressing cells, another downstream effector of Flt3. In an effort to determine if Flt3Va+ cells had tumor repopulating ability, we sorted 0.3X10^6 Flt3Va+ and Flt3Va- cells from a PDX sample and injected the sorted populations or unsorted bulk tumor cells into NSG mice. The human cell engraftment in the mice was detected by the expression of human CD45, CD33, CD34, CD38, and c-kit antigens in the peripheral blood. In two experiments, mice injected with Flt3Va+ cells had detectable circulating leukemic cells by ~18 days after injection, while those injected with Flt3Va- cells had detectable circulating leukemic cells after the 4th week. These results suggest both Flt3Va+ and Flt3Va- cell populations are able to reconstitute leukemia after transplantation in NSG mice. However, Flt3Va+ may be expressed by an aggressive AML clone that facilitate early tumor engraftment. Overall, these studies suggest that Flt3Va is a leukemia-specific neoantigen and is an attractive potential immunotherapeutic target in AML. Proteins such as Flt3Va generated by alternative splicing are common in AML and may be targets for of novel blocking antibodies or ADCs, minimizing effects on normal tissues. Disclosures Adamia: Janssen: Research Funding. Nemeth:Janssen: Employment. Attar:Janssen: Employment. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding. Steensma:Millenium/Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Ariad: Equity Ownership; Genoptix: Consultancy. Weinstock:Novartis: Consultancy, Research Funding. DeAngelo:Novartis: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; Baxter: Consultancy; Celgene: Consultancy; Incyte: Consultancy; Amgen: Consultancy. Stone:Agios: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celator: Consultancy; Juno Therapeutics: Consultancy; Roche: Consultancy; Jansen: Consultancy; Pfizer: Consultancy; ONO: Consultancy; Sunesis Pharmaceuticals: Consultancy; Merck: Consultancy; Xenetic Biosciences: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Amgen: Consultancy; Karyopharm: Consultancy; Seattle Genetics: Consultancy. Griffin:Janssen: Research Funding; Novartis: Consultancy, Research Funding.

Description: Interleukin (IL)-12 plays a critical role in modulating the activities of natural killer (NK) cells and T lymphocytes. In animal models, IL-12 has potent antitumor effects that are likely mediated by its ability to enhance the cytotoxic activity of NK cells and cytotoxic T lymphocytes, and to induce the production of interferon (IFN)-γ by NK and T cells. In addition to IL-12, NK cells are responsive to IL-2, and may mediate some of the antitumor effects of IL-2. In this study, we examine the interaction between IL-2 and the signaling events induced by IL-12 in NK cells. We find that IL-2 not only up-regulates the expression of IL-12Rβ1 and IL-12Rβ2, it also plays an important role in up-regulating and maintaining the expression of STAT4, a critical STAT protein involved in IL-12 signaling in NK cells. In contrast to the effects of IL-2 alone, expression of IL-12 receptors and STAT4 are unaffected or decreased by IL-12 or the combination of IL-2 and IL-12. Through expression of high levels of IL-12 receptors and STAT4, IL-2–primed NK cells show enhanced functional responses to IL-12 as measured by IFN-γ production and the killing of target cells. NK cells from cancer patients who received low-dose IL-2 treatment also exhibited increased expression of IL-12 receptor chains, suggesting that IL-2 may enhance the response to IL-12 in vivo. These findings provide a molecular framework to understand the interaction between IL-2 and IL-12 in NK cells, and suggest strategies for improving the effectiveness of these cytokines in the immunotherapy of cancer.

Description: Interleukin (IL)-12 plays a critical role in modulating the activities of natural killer (NK) cells and T lymphocytes. In animal models, IL-12 has potent antitumor effects that are likely mediated by its ability to enhance the cytotoxic activity of NK cells and cytotoxic T lymphocytes, and to induce the production of interferon (IFN)-γ by NK and T cells. In addition to IL-12, NK cells are responsive to IL-2, and may mediate some of the antitumor effects of IL-2. In this study, we examine the interaction between IL-2 and the signaling events induced by IL-12 in NK cells. We find that IL-2 not only up-regulates the expression of IL-12Rβ1 and IL-12Rβ2, it also plays an important role in up-regulating and maintaining the expression of STAT4, a critical STAT protein involved in IL-12 signaling in NK cells. In contrast to the effects of IL-2 alone, expression of IL-12 receptors and STAT4 are unaffected or decreased by IL-12 or the combination of IL-2 and IL-12. Through expression of high levels of IL-12 receptors and STAT4, IL-2–primed NK cells show enhanced functional responses to IL-12 as measured by IFN-γ production and the killing of target cells. NK cells from cancer patients who received low-dose IL-2 treatment also exhibited increased expression of IL-12 receptor chains, suggesting that IL-2 may enhance the response to IL-12 in vivo. These findings provide a molecular framework to understand the interaction between IL-2 and IL-12 in NK cells, and suggest strategies for improving the effectiveness of these cytokines in the immunotherapy of cancer.