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: T-cell activation in response to interleukin-12 (IL-12) is mediated through signaling events that include the tyrosine phosphorylation of STAT4. IL-12 responsiveness and the ability of IL-12 to activate STAT4 is different in T cells induced to differentiate into a Th1 or Th2 phenotype. In this report, we show that STAT5, STAT1α, and STAT1β, in addition to STAT4, are tyrosine phosphorylated in response to IL-12 in phytohemagglutinin (PHA)-activated human T cells. To understand how the activation of these STATs contributes to T-cell IL-12 responsiveness, we analyzed the IL-12–induced activation of STAT5 and STAT1 in T cells stimulated to undergo Th1 or Th2 differentiation. The IL-12–induced tyrosine phosphorylation of STAT5 and STAT1, but not STAT4, is augmented in T cells activated into Th1 cells with PHA + interferon-γ (IFN-γ) compared with T cells activated with PHA alone. STAT5 DNA binding induced by IL-12 is also augmented in T cells activated with PHA + IFN-γ compared with T cells activated with PHA alone, whereas STAT4 DNA binding is not increased. In contrast, the IL-12–induced activation of these STATs is inhibited in T cells activated into Th2 cells with PHA + IL-4. The enhancement of IL-12 signaling by IFN-γ is not a direct effect of IFN-γ on T cells, but rather is mediated by IL-12 that is produced by antigen-presenting cells in response to IFN-γ. This positive autoregulatory effect of IL-12 on the activation of select STATs correlates with an increase in T-cell IFN-γ production in response to IL-12. These findings suggest that the activation of STAT5 and STAT1 may augment select STAT4-dependent functional responses to IL-12 in Th1 cells.

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: To study constitutive Janus kinase signaling, chimeric proteins were generated between the pointed domain of the etstranscription factor TEL and the cytosolic tyrosine kinase Jak2. The effects of these proteins on interleukin-3 (IL-3)–dependent proliferation of the hematopoietic cell line, Ba/F3, were studied. Fusion of TEL to the functional kinase (JH1) domain of Jak2 resulted in conversion of Ba/F3 cells to factor-independence. Importantly, fusion of TEL to the Jak2 pseudokinase (JH2) domain or a kinase-inactive Jak2 JH1 domain had no effect on IL-3–dependent proliferation of Ba/F3 cells. Active TEL-Jak2 constructs (consisting of either Jak2 JH1 or Jak2 JH2+JH1 domain fusions) were constitutively tyrosine-phosphorylated but did not affect phosphorylation of endogeneous Jak1, Jak2, or Jak3. TEL-Jak2 activation resulted in the constitutive tyrosine phosphorylation of Stat1, Stat3, and Stat5 as determined by detection of phosphorylation using activation-specific antibodies and by binding of each protein to a preferential GAS sequence in electrophoretic mobility shift assays. Elucidation of signaling events downstream of TEL-Jak2 activation may provide insight into the mechanism of leukemogenesis mediated by this oncogenic fusion protein.

Description: Acute Myeloid Leukemia (AML) and Myelodysplastic syndrome (MDS) arise from accumulation of multiple stepwise genetic and epigenetic changes in hematopoietic stem cells (HSC) and/or committed progenitors. A series of transforming events can initially give rise to pre-leukemia stem cells (pre-LSC) as well as fully transformed leukemia stem cells (LSC), both of which need to be targeted in strategies aimed at curing these diseases. We conducted parallel transcriptional and epigenetic analysis of highly fractionated stem and progenitor populations in individual patients of MDS and identified STAT3 upregulation in MDS HSCs. qRTPCR in an independent set of sorted MDS/AML HSCs (Lineage-negative, CD34+, CD38-) confirmed the significant increase in STAT3 expression in 60% of cases when compared to healthy controls. We further analyzed gene expression profiles of CD34+ cells from 183 MDS patients and found significant increased expression of STAT3 in MDS when compared to healthy controls (FDR