ALBERT

Beschreibung: 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..

Beschreibung: 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.

Beschreibung: 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)

Beschreibung: 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

Beschreibung: Abstract 399 Introduction: The transcription factor STAT5 is constitutively activated in many forms of hematologic malignancies, including chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). STAT5 can be activated by constitutively activated tyrosine kinases or autocrine and paracrine secretion of cytokines signaling through Jak kinases. STAT5 is essential for the pathogenesis of neoplasms induced by BCR-ABL1 and Jak2V617F, as well as for leukemia stem cell self-renewal. Development of tyrosine kinases inhibitors (TKIs), such as imatinib, has greatly improved the outcome of patients with leukemias harboring aberrantly activated oncogenic tyrosine kinases. However, TKIs used as a single agent only achieve significant success in CML, with very limited benefit in the more aggressive ALL. Moreover, patients with CML who initially respond well may acquire resistance to TKIs with the progression of their disease. In fact, increased activity of STAT5 is often associated with CML progression and may underlie resistance to TKIs. Importantly, leukemia cells that are resistant to TKIs remain sensitive to STAT5 inhibition, and dual inhibition of both tyrosine kinases and STAT5 leads to more efficient reduction of leukemia cell viability. Thus targeting STAT5 alone or in combination is a promising therapeutic strategy for many hematological malignancies. While many strategies directly inhibit STAT5, we considered the possibility that STAT5 association with co-regulatory proteins is essential for STAT5 function and therefore targeting this association may be a suitable therapeutic strategy. Methods and Results: Given the importance of BET bromodomain proteins in chromatin remodeling necessary for transcription, we tested the activity of the BET bromodomain inhibitor JQ1 on STAT5-dependent transcriptional activity. Using both heterologous reporter systems and endogenous STAT5 target genes, we found that JQ1, but not its inactive enantiomer, potently and specifically inhibited STAT5-dependent gene expression. Inhibition of STAT5 dependent gene regulation was also replicated by another BET bromodomian inhibitor, iBET, further demonstrating that BET inhibition inhibits STAT5. Since JQ1 inhibits BET family members Brd2, Brd3, Brd4, and BrdT, we asked which BET family member is specifically associated with STAT5 transcriptional function. To do this, we utilized shRNA to knock-down each bromodomain protein and determined the effect on STAT5 activity. We found that knocking-down Brd2, but not Brd3 or Brd4, reduces STAT5 target gene expression, indicating that Brd2 is specifically involved in regulating STAT5 transcriptional function. JQ1 can reduce STAT5 transcriptional activity without inhibiting STAT5 phosphorylation or STAT5 binding to its genomic binding sites. Similarly, knocking-down Brd2 can reduce STAT5 target gene expression without influencing STAT5 phosphorylation. We hypothesize that Brd2 regulates STAT5 transcriptional function by acting as a co-activator for STAT5. Thus through blocking Brd2, JQ1 can inhibit STAT5 transcriptional function without directly targeting STAT5 itself. In a group of aggressive T cell acute lymphoblastic leukemia (T-ALL) cell lines, where constitutively activated STAT5 contributes to leukemia cell survival, knocking-down Brd2 renders leukemia cells more sensitive to TKI induced apoptosis. In addition, combined treatment with TKIs and JQ1 showed strong synergy in inducing T-ALL leukemia cells apoptosis and reducing viability. Overexpressing a constitutively active form of STAT5 rescues these leukemia cells from death induced by TKIs and JQ1, indicating an important role of STAT5 as a target for TKI and JQ1 induced cell death in T-ALL cells. Conclusion: We found that the BET bromodomain inhibitor JQ1 can reduce STAT5 transcriptional function by blocking Brd2 without reducing STAT5 phosphorylation or STAT5 DNA binding. In addition, the combination of TKIs and JQ1 induces T-ALL leukemia cell apoptosis and reduces survival in a synergistic manner, and represents a rational drug combination for treating this sub-group of highly aggressive leukemias. Disclosures: Bradner: Tensha Therapeutics: Consultancy, Equity Ownership, Scientific founder Other.

Beschreibung: Chronic myelogenous leukemia (CML) is characterized by the BCR/ABL fusion tyrosine kinase which mediates its oncogenic effects in part through constitutive activation of the transcription factor STAT5. BCR/ABL can be inhibited by several kinase inhibitors, including imatinib, which leads to apoptosis of the cells. However, the development of resistance to these agents, most commonly mediated through point mutations in the kinase, is an increasing problem. Since STAT5 is a critical mediator of the effects of BCR/ABL, the development of drugs inhibiting this transcription factor holds promise as an independent means to inhibit BCR/ABL-transformed cells. Therefore, we developed a high throughput cell-based screen to identify drugs that specifically inhibit the transcriptional function of STAT proteins. To accelerate the introduction of active agents into clinical trials, we have focused on drugs that are approved for use, or are otherwise known to be safe, in humans. Using this approach, we have identified pimozide, a neuroleptic drug, as an inhibitor of STAT5. Pimozide decreases STAT5 phosphorylation in CML cells but, in contrast to imatinib, it does not inhibit BCR/ABL kinase activity. Furthermore, pimozide decreases expression of STAT5 target genes in these cells. Consistent with the known role of STAT5 in promoting the survival and proliferation of BCR/ABL-transformed cells, pimozide induces apoptosis in the K562 CML cell line. At similar concentrations, pimozide induces little toxicity in peripheral blood mononuclear cells from normal donors. Given the clinical importance of point mutations in BCR/ABL in the development of resistance to tyrosine kinase inhibitors, we evaluated the effects of pimozide in Ba/f3 cells reconstituted with wildtype BCR/ABL or BCR/ABL containing a T315I mutation, which is highly resistant to current kinase inhibitors. Pimozide reduces the viability of both cell types with similar dose response, providing further support to the hypothesis that this drug works by a mechanism distinct from imatinib. Given this, we considered the possibility that the combination of pimozide and imatinib might be more effective than either drug alone. Treatment of KU812 CML cells with low concentrations of both pimozide and imatinib induced greater loss of viability than either drug alone, with characteristics consistent with pharmacological synergy. In addition, combination treatment with pimozide and imatinib induces a greater level of apoptosis in CML cells than either drug alone. In conclusion, we have identified pimozide as a STAT5 inhibitor that is effective at reducing the viability of CML cells, including those resistant to imatinib. This suggests that the identification of inhibitors of STAT transcription factors may be a promising method for developing new anti-cancer therapies. In addition, the use of pimozide alone or in conjunction with kinase inhibitors may be an important new strategy for the treatment of CML or other diseases characterized by constitutive STAT activation.

Beschreibung: IL-2 plays a critical role in the maintenance of CD4+CD25+ FOXP3+ regulatory T cells (Tregs) in vivo. We examined the effects of IL-2 signaling in human Tregs. In vitro, IL-2 selectively up-regulated the expression of FOXP3 in purified CD4+CD25+ T cells but not in CD4+CD25- cells. This regulation involved the binding of STAT3 and STAT5 proteins to a highly conserved STAT-binding site located in the first intron of the FOXP3 gene. We also examined the effects of low-dose IL-2 treatment in 12 patients with metastatic cancer and 9 patients with chronic myelogenous leukemia after allogeneic hematopoietic stem cell transplantation. Overall, IL-2 treatment resulted in a 1.9 median fold increase in the frequency of CD4+CD25+ cells in peripheral blood as well as a 9.7 median fold increase in FOXP3 expression in CD3+ T cells. CD56+CD3- natural killer (NK) cells also expanded during IL-2 therapy but did not express FOXP3. In vitro treatment of NK cells with 5-aza-2′-deoxycytidine restored the IL-2 signaling pathway leading to FOXP3 expression, suggesting that this gene was constitutively repressed by DNA methylation in these cells. Our findings support the clinical evaluation of low-dose IL-2 to selectively modulate CD4+CD25+ Tregs and increase expression of FOXP3 in vivo.

Beschreibung: The transcription factor STAT5 is an essential mediator of the pathogenesis of chronic myelogenous leukemia (CML). In CML, the BCR/ABL fusion kinase causes the constitutive activation of STAT5, thereby driving the expression of genes promoting survival. BCR/ABL kinase inhibitors have become the mainstay of therapy for CML, although CML cells can develop resistance through mutations in BCR/ABL. To overcome this problem, we used a cell-based screen to identify drugs that inhibit STAT-dependent gene expression. Using this approach, we identified the psychotropic drug pimozide as a STAT5 inhibitor. Pimozide decreases STAT5 tyrosine phosphorylation, although it does not inhibit BCR/ABL or other tyrosine kinases. Furthermore, pimozide decreases the expression of STAT5 target genes and induces cell cycle arrest and apoptosis in CML cell lines. Pimozide also selectively inhibits colony formation of CD34+ bone marrow cells from CML patients. Importantly, pimozide induces similar effects in the presence of the T315I BCR/ABL mutation that renders the kinase resistant to presently available inhibitors. Simultaneously inhibiting STAT5 with pimozide and the kinase inhibitors imatinib or nilotinib shows enhanced effects in inhibiting STAT5 phosphorylation and in inducing apoptosis. Thus, targeting STAT5 may be an effective strategy for the treatment of CML and other myeloproliferative diseases.

Beschreibung: Constitutive activation of the transcription factor STAT3 contributes to the pathogenesis of many cancers, including multiple myeloma (MM). Since STAT3 is dispensable in most normal tissue, targeted inhibition of STAT3 is an attractive therapy for patients with these cancers. To identify STAT3 inhibitors, we developed a transcriptionally based assay and screened a library of compounds known to be safe in humans. We found the drug nifuroxazide to be an effective inhibitor of STAT3 function. Nifuroxazide inhibits the constitutive phosphorylation of STAT3 in MM cells by reducing Jak kinase autophosphorylation, and leads to down-regulation of the STAT3 target gene Mcl-1. Nifuroxazide causes a decrease in viability of primary myeloma cells and myeloma cell lines containing STAT3 activation, but not normal peripheral blood mononuclear cells. Although bone marrow stromal cells provide survival signals to myeloma cells, nifuroxazide can overcome this survival advantage. Reflecting the interaction of STAT3 with other cellular pathways, nifuroxazide shows enhanced cytotoxicity when combined with either the histone deacetylase inhibitor depsipeptide or the MEK inhibitor UO126. Therefore, using a mechanistic-based screen, we identified the clinically relevant drug nifuroxazide as a potent inhibitor of STAT signaling that shows cytotoxicity against myeloma cells that depend on STAT3 for survival.