Apoptosis In CLL Research Articles

Using NK-Derived Exosomes to Treat Leukemia

Natural killer cells (NK) are components of the innate immune system which play an important role in surveillance of cancer cells. Despite promising results in clinical trials, the use of NK-based therapies is limited due to safety issues. Exosomes are nanosized extracellular vesicles secreted by all types of cells. In recent years, exosomes have emerged as a powerful, natural therapeutic tool due to their low immunogenicity, low cytotoxicity, prolonged bioavailability and superior targeting ability. Since exosomes are known to carry cargo that reflects their cell of origin, we were prompted to test whether NK-derived exosomes (NK exo) maintain the anti-leukemia capacity of NK cells. To test the feasibility of this approach we undertook a set of preliminary experiments and evaluated the anti-leukemic potential of NK exo in-vitro on a wide variety of leukemic cell lines and patient-derived samples while also striving to assess their killing potential in-vivo using a murine xenograft model . This study aims to provide the pre-clinical evidence needed to test the NKexo approach in future clinical studies with the intention of ultimately developing an acellular “off-the-shelf” product to treat leukemia.Initially, we isolated NK exo from the engineered NK92MI cell line by ultracentrifugation and demonstrated the presence of large amounts of 100-200nm cap-shaped particles by means of nano-tracking-analysis and transmission-electron-microscopy. We then showed that these particles express the exosomal-characteristic CD63- and CD81- and NK-characteristic CD56-biomarkers. Next, we evaluated the effect of NK exo on CML (K562), T-ALL (Jurkat), B-ALL (UoC) and AML (HL-60 and KG1a) cell lines. NK exo treatment initiated a time- and dose-dependent reduction in viability and induction of apoptosis in all cell lines tested. The effect of NK exo exposure on cell viability and apoptosis was reduced in the presence of the pan-caspase inhibitor Z-VAD-FMK suggesting that NK exo exert their cytotoxic effect in a caspase-dependent manner. In addition, exposure to NK exo led to a significant increase in apoptosis in treatment naive AML, CLL and ALL patient-derived mononuclear cells. Moreover, we witnessed a significant reduction in colony formation of mononuclear cells harvested from treatment naive CML and AML patients in the presence of NK exo. While leukemia cells were targeted and severely affected by NK exo, healthy B-cells remained unaffected, indicating a selective effect. The selective trait of NK exo was also confirmed via an exosome uptake assay which demonstrated that NK exo were taken up specifically by leukemic cells but not by healthy B-cells.In an in-vivo xenograft model for AML; NOD-SCID IL2Rgama null (NSG) mice were engrafted with the well characterized human AML cell line HL60, and were then intravenously injected with NK exo. Our preliminary results indicate that NK exo treatment improves overall mice survival indicating an anti-leukemic effect of NK exo in-vivo.In this “proof-of-concept” study, we show that NK exo, similar to their cell-of-origin, have a strong and selective anti-leukemia effect. To provide the pre-clinical evidence needed to test the NK exo approach in future clinical studies, we are currently continuing to test the anti-leukemic potential of NK exo in-vivo on a xenograft murine model. Our preliminary results propose the use of NK exo as a novel therapeutic platform to combat leukemia. More broadly, since NK cells target a wide range of cancer cells, not just leukemia cells, and since we have shown that NK exo maintain the anti-leukemic capacity of their donor cells, this approach may become applicable in other hematological and solid malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Very Early Effects of Ibrutinib on Tumor and Immune Cells in Blood and Lymph Nodes in Relapsed or Refractory Chronic Lymphocytic Leukemia (CLL) Patients

In this study we analyzed the very early effects of the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib on tumor and immune cells in 7 symptomatic, relapsed or refractory CLL patients during the first four weeks of treatment. Five of the patients had Rai stage IV at study entry and four patients had 17p deletion or TP53 mutation. Median number of previous treatment regimens was 2 (range 1-4). Peripheral blood (PB) samples were collected before (< 1 week) treatment start and at six different time points during the treatment (9 hours after treatment start on day 1; on day 2; day 4; day 8; day 15 and day 29). Fine needle aspiration of two pathological lymph nodes (LN) identified by ultrasound was performed before (< 1 week) treatment start and at day 2, day 8 and day 29. Flow-cytometry was performed to analyze the changes in the peripheral blood mononuclear cell populations in PB including CLL cells, Natural Killer (NK) cells, monocytes, dendritic cells (DC) and T cells memory subsets, helper subpopulations (Ths) and regulatory T cells (Tregs). Moreover, changes in expression of 18 B-cell activation and migration markers on CLL cells as well as T-cell activation and proliferation markers, were analyzed in paired LN and PB samples. Finally, plasma levels of 92 inflammation-related protein biomarkers were assessed by Multiplex Proximity Extension Assay (PEA).In six out of seven patients the size of the LN decreased gradually during the four weeks of observation, achieving complete clinical remission in three patients and partial remission in three other. In 5 evaluated patients, the CLL cell counts in PB increased already 9 hours after treatment start (fold increase 1.36-2.84). In all the patients, CLL cells were higher at day 2 and 8 compared to baseline (p=0.02), and decreased by day 29 to levels not significantly different from the baseline. Over the four weeks period CD4+ cells increased (p=0.03), while CD8+, NK, and NKT cells remained stable. The distribution of the CD4+ and CD8+ memory cell subsets remained unchanged. Th1 cells increased (p=0.01) while Th2, Th17 and Tregs were stable.Expression of Programmed cell death protein 1 (PD-1) and Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) in T cells was unaffected. No effect was seen on CLL apoptosis. Proliferating (Ki67+) CLL cells, which at baseline varied between 1.2-10.8%, were not detected in blood after 1 week of treatment (p=0.0006). Ki67+ T cells were also eliminated (p=0.0007), but with a delayed kinetics (4 weeks).Paired LN and PB samples were screened for changes in 18 surface markers relevant for B-cell activation and migration. With the exception of CD23, whose expression was lower in PB compared to LN (p=0.02), no difference was observed in the expression of the other analyzed surface markers on CLL cells from the two different tumor compartments at baseline. CD5 expression remained stable during treatment, albeit decreasing significantly by day 29 in PB (p=0.02). Expression of CD20 decreased in both compartments at day 8 (p=0.02) and CD40 decreased at day 8 only in PB (p=0.02). CD69 expression (MFI) decreased both in PB and LN by day 8 (p=0.02), while the percentage of positive cells significantly dropped already at day 2 in PB (p=0.02) and from day 8 in LN (p=0.03). CD23 MFI decreased in the LN compartment already at day 2 (p=0.02), but the percentage of positive cells was significantly lower only at day 8 (p=0.03). Compared to LN, CD23 expression was lower in PB before treatment and therefore the significant drop both in MFI and percentage of positive cells was seen only after 4 weeks (p=0.02). No change in the expression of CD49d was observed.In 5 evaluable patients, the plasmacytoid DCs, undetectable at baseline, increased during treatment (p=0.06), while CD16+SLAN+monocytes decreased (p=0.06).Finally, plasma levels of 50 molecules were significantly changed at ≥ 1 time point during treatment. With the exception of CST5 and IL-17C, which were increased, the residual 48 proteins were all down-regulated, some of them (e.g. CCL3 and CCL4) already by 9 hours. The majority of the cytokines with significantly reduced levels are pro-inflammatory molecules.In conclusion, these data indicate that ibrutinib causes major changes both in CLL and bystander cells as well as in the levels of several inflammation-related protein biomarkers already shortly after treatment initiation. DisclosuresLundin:Janssen: Research Funding. Kimby:Gilead: Honoraria, Other: honoraria for educational lecture in meeting sponsored by Gilead; Roche: Other: Honoraria for lecture in educational meetings; Pfizer: Other: Research grant; Celgene: Other: Honoraria for lecture. educational meeting; Jansen: Membership on an entity’s Board of Directors or advisory committees, Other: Honoraria for lecture at educational session. Österborg:Janssen: Honoraria, Research Funding.

Inhibition of JAK2/STAT3 Pathway Leads to Apoptosis in Chronic Lymphocytic Leukemia Cells

INTRODUCTIONChronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature clonal CD19+/CD5+/CD23+ B lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. Despite their in vivo prolonged lifespan due to intrinsic defects, CLL leukemic cells rapidly undergo spontaneous apoptosis in vitro, highlighting the need of extrinsic signals delivered by the microenvironment.Several molecules, including those released by mesenchymal stromal cells (MSCs), signal through JAK (Janus kinases)-STAT (Signal Transducers and Activators of Transcription) pathways. We particularly focused on the JAK2/STAT3 axis since Interleukin-6 (IL-6), one of the most abundant cytokines released in the CLL microenvironment, is the key ligand of the receptor triggering this pathway. The deregulation of JAK2/STAT3 axis may lead to aberrant activation of STAT3 and, as a result, to tumor development in hematopoietic cells.METHODSB cells were collected from 12 controls and 46 CLL patients. Purified cells (2x106cells/ml) were cultured, and treated with AG490 (10, 50 and 100μM), AZD1480 (1, 4 and 10μM), Fedratinib (1, 5 and 10μM), and Ruxolitinib (0.313, 2.5 and 10μM) (which are JAK2 inhibitors), and the STAT3 inhibitor Stattic (5, 7.5, and 10μM) for 24, 48 and 72h. Experiments with AG490 and Stattic were performed with/without MSCs.STAT3 expression and phosphorylation were evaluated by Western Blotting (WB) and Flow Cytometry (FC), and its localization was analyzed by confocal microscopy and subcellular fractionation. CLL and normal B cell viability was tested by FC with Annexin V/PI test.RESULTSWe demonstrated that STAT3 was highly expressed in malignant B cells with respect to normal B lymphocytes. As far as STAT3 phosphorylation at Tyr705, that is an essential step for STAT3 activation, we demonstrated a constitutive phosphorylation in CLL cells by FC and WB analyses, although in some patients STAT3 Tyr705 phosphorylation is barely detected.We also pointed out that the in vitroincubation of leukemic B cells with AG490 and Stattic and Fedratinib, induces a dose-dependent apoptosis of CLL B cells. However, the tested doses of Ruxolitinib and AZD1480 did not seem to CLL B cell viability but only STAT3 phosphorylation. Both AG490 and Stattic were able to bypass the microenvironmental protection when neoplastic B cells were co-cultured with MSCs.STAT3 Tyr705 localization was analyzed in normal and leukemic B cells by a subcellular protein fractionation. We separated nuclei from cytosol, detecting STAT3 Tyr705 both in the cytosolic and in the nuclear fractions of CLL B cells.We showed that AG490 and Fedratinib treatment on CLL cells can mediate other effects: i) SHP-1 activity is turned on by JAK2 inhibition, decreasing its phosphorylation at Ser591; ii) AG490 administration inactivates protein Lyn, reducing the phosphorylation in its active site at Tyr396.Lyn, a Tyr-kinase, and SH2-domain containing Tyrosine Phosphatase (SHP-1), a Tyr-phosphatase, are both involved in the prolonged lifespan of neoplastic CLL cells. To confirm the link between JAK2 inhibition by AG490 and Lyn dephosphorylation, we added sodium orthovanadate (Na3VO4, 100 μM), a phosphatase inhibitor, to cell culture to restore Lyn activation (resulting from the inactivation of SHP-1 phosphatase); as a result, Lyn Tyr396 phosphorylation was restored. On the contrary, the treatment of CLL cells with Stattic did not induce any change in SHP-1 status with respect to untreated cells since Stattic is effective on STAT3, that is a downstream protein with respect to JAK2. Since Stattic did not affect SHP-1 activation, it does not impact on Lyn activation/phosphorylation.CONCLUSIONSThe ability of AG490 and Stattic to induce apoptosis in leukemic B cells bypassing the pro-survival stimuli provided by the tumor microenvironment and the Fedratinib effectiveness at low doses, represents a starting point for the development of new therapeutic strategies in CLL.This study also provides new insights for the investigation of the pathogenesis of CLL focusing the attention on the cross-talk between JAK/STAT and BCR/Lyn axes. DisclosuresNo relevant conflicts of interest to declare.

The isopeptidase inhibitor 2cPE triggers proteotoxic stress and ATM activation in chronic lymphocytic leukemia cells.

Relapse after treatment is a common and unresolved problem for patients suffering of the B-cell chronic lymphocytic leukemia (B-CLL). Here we investigated the ability of the isopeptidase inhibitor 2cPE to trigger apoptosis in leukemia cells in comparison with bortezomib, another inhibitor of the ubiquitin-proteasome system (UPS). Both inhibitors trigger apoptosis in CLL B cells and gene expression profiles studies denoted how a substantial part of genes up-regulated by these compounds are elements of adaptive responses, aimed to sustain cell survival. 2cPE treatment elicits the up-regulation of chaperones, proteasomal subunits and elements of the anti-oxidant response. Selective inhibition of these responses augments apoptosis in response to 2cPE treatment. We have also observed that the product of the ataxia telangiectasia mutated gene (ATM) is activated in 2cPE treated cells. Stimulation of ATM signaling is possibly dependent on the alteration of the redox homeostasis. Importantly ATM inhibition, mutations or down-modulation increase cell death in response to 2cPE. Overall this work suggests that 2cPE could offer new opportunities for the treatment of B-CLL.

Abstract B02: The Akt inhibitor MK-2206 in combination with rituximab and bendamustine demonstrates efficacy in relapsed/refractory chronic lymphocytic leukemia: Updated results from the NCCTG N1087 Alliance study

Abstract Background: PI3K/Akt activation is downstream of the B cell receptor (BCR) signaling cascade and is critical to mediate the interactions between CLL B-cell and bone marrow stroma which support leukemic survival. MK-2206 is a potent oral allosteric Akt inhibitor and we have demonstrated in vitro synergism with bendamustine (B) to induce CLL apoptosis. MK-2206 selectively abolishes BCR-stimulated cytokines CCL3, CCL4, CCL2, and IL-2Ra and significantly inhibits the BCR signaling pathway (Ding, BJH, 2013). We sought to assess the safety, maximal tolerated dose (MTD) and efficacy of MK-2206 in combination with B-rituximab (BR) in relapsed and refractory CLL/SLL patients in a phase I/II trial (NCCTG N1087 Alliance). Methods: Previously treated symptomatic CLL/SLL patients with up to 3 prior lines of therapy and ECOG performance status of 0-2 were eligible. Patients with 17p deletion or prior treatment with B were excluded. A standard phase I design was used to determine the MTD of MK-2206 in combination with BR (B 70mg/m2 for 2 days per cycle; R cycle 1: 375 mg/m2, cycle 2-6: 500 mg/m2). Phase II employed the MTD to evaluate safety and efficacy of the combination. Response was evaluated 2 months after the last cycle of therapy per IWCLL 2008 criteria. Results: 13 patients with a median age of 68 years (range 44-75) and 1.2 prior lines of therapy were treated. CLL FISH showed: del(11q) in 4 (31%), trisomy 12 in 2 (15%), del(13q) in 3 (23%), del(6q) (8%), del(17cen) (8%) and normal results in 2 (15%). IGHV was unmutated in 10 (91%) and mutated in 1 patient (9%) and was missing in 2 cases. ZAP-70 was positive in 8 (73%) and negative in 3 patients (27%). CD38 was positive in 8 (62%) and negative in 5 patients (38%). 70% of patients had received prior chemoimmunotherapy. At 90 mg of MK-2206, 1/6 patients experienced a DLT of grade 3 febrile neutropenia and hemolysis. 2/6 patients on the 135 mg dose experienced a DLT. One patient had grade 3 febrile neutropenia and one patient had a grade 3 acneiform rash. MTD was determined to be MK-2206 90 mg. The most common grade 3/4 adverse events were neutropenia (46%) including febrile neutropenia (23%), diarrhea (15%) and thrombocytopenia (15%). The most common all-grade toxicities were neutropenia (69%), thrombocytopenia (62%), anemia (54%), nausea (54%), diarrhea (39%), rash (38%), and hyperglycemia (31%). 10 patients were treated at 90 mg and 3 patients were treated at the 135 mg dose. The ORR was 92% (n=12). Responses were: 3 CR (23%), 2 CR with incomplete marrow recovery (CRi) (15%), 1 clinical CR (CCR) (8%) without marrow confirmation, 1 nodular partial remission (nPR) (8%), 5 PR (38%), and 1 progressive disease (8%). Median follow-up was 20 months (6-31 months). Median PFS in the CR/CRi group was not reached (NR) with 100% of patients progression free versus 12 months (95% CI 2 months-NR) if CR/CRi was not achieved (p=0.027). Median overall survival was NR in either the CR/CRi or non-CR/CRi groups but 2 deaths occurred in the latter group (p=0.44). One patient developed Richter transformation and died at 15 months and a second patient who had achieved PR died from autoimmune hemolytic anemia and fungal infection at 27 months. Two out of five patients who achieved CR or CRi had bone marrow MRD negative status at the final response evaluation. Conclusions: The Akt inhibitor MK-2206 administered at 90 mg once weekly in combination with BR is tolerated in patients with relapsed or refractory CLL and compares favorably to BR alone (59% ORR and 9% CR, Fischer, JCO, 2011). An ORR of 92% was observed with a 38% of patients achieving CR or CRi. The trial was terminated prematurely due to withdrawal of sponsor support, however further testing of Akt inhibition is needed given the promising results. Citation Format: Jeremy T. Larsen, Tait D. Shanafelt, Jose F. Leis, Betsy R. LaPlant, Timothy G. Call, Clive S. Zent, Curtis A. Hanson, Charles Erlichman, Thomas M. Habermann, Craig B. Reeder, Deborah A. Bowen, Michael Conte, Justin C. Boysen, Charla R. Secreto, Connie E. Lesnick, Renee C. Tschumper, Diane F. Jelinek, Neil E. Kay, Wei Ding. The Akt inhibitor MK-2206 in combination with rituximab and bendamustine demonstrates efficacy in relapsed/refractory chronic lymphocytic leukemia: Updated results from the NCCTG N1087 Alliance study. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B02.

Viability and stress protection of chronic lymphoid leukemia cells involves overactivation of mitochondrial phosphoSTAT3Ser727.

Chronic lymphoid leukemia (CLL) is characterized by the accumulation of functionally defective CD5-positive B lymphocytes. The clinical course of CLL is highly variable, ranging from a long-lasting indolent disease to an unpredictable and rapidly progressing leukemia requiring treatment. It is thus important to identify novel factors that reflect disease progression or contribute to its assessment. Here, we report on a novel STAT3-mediated pathway that characterizes CLL B cells-extended viability and oxidative stress control. We observed that leukemic but not normal B cells from CLL patients exhibit constitutive activation of an atypical form of the STAT3 signaling factor, phosphorylated on serine 727 (Ser727) in the absence of detectable canonical tyrosine 705 (Tyr705)-dependent activation in vivo. The Ser727-phosphorylated STAT3 molecule (pSTAT3Ser727) is localized to the mitochondria and associates with complex I of the respiratory chain. This pSer727 modification is further controlled by glutathione-dependent antioxidant pathway(s) that mediate stromal protection of the leukemic B cells and regulate their viability. Importantly, pSTAT3Ser727, but neither Tyr705-phosphorylated STAT3 nor total STAT3, levels correlate with prolonged in vivo CLL B cells survival. Furthermore, STAT3 activity contributes to the resistance to apoptosis of CLL, but not normal B cells, in vitro. These data reveal that mitochondrial (Mt) pSTAT3Ser727 overactivity is part of the antioxidant defense pathway of CLL B cells that regulates their viability. Mt pSTAT3Ser727 appears to be a newly identified cell-protective signal involved in CLL cells survival. Targeting pSTAT3Ser727 could be a promising new therapeutic approach.

The AKT Inhibitor MK2206 In Combination With Rituximab and Bendamustine Is Tolerable and Active In Relapsed Or Refractory Chronic Lymphocytic Leukemia: Results From a Phase 1 Study (NCCTG N1087 Alliance)

BackgroundsB cell receptor (BCR) signals and microenvironment protection have been recognized to have key influences on CLL survival and activation. Akt activation is down-stream of the BCR signaling cascade and is critical in the mediation of the bi-directional interactions between CLL B-cells and bone marrow stroma. MK2206 is an allosteric Akt inhibitor previously tested for safety in solid tumor patients. Our preclinical data has demonstrated the synergy of MK2206 with bendamustine (B) to induce CLL apoptosis in vitro. MK2206 also abolished the signal activation and cytokine production induced by BCR ligation in CLL leukemic cells (Ding, ASH 2012). Therefore, we conducted a phase 1 trial testing the safety and efficacy of MK2206 in combination with bendamustine and rituximab (BR) for patients with relapsed/refractory CLL. MethodsA phase 1 dose-escalation study was designed to define the maximum tolerated dose (MTD) as well as the safety and efficacy of 3 dose levels (90 mg or 135 mg or 200 mg weekly) of the oral Akt inhibitor MK2206 in combination with bendamustine (70 mg/m2 daily for 2 days in each cycle) and rituximab (cycle 1: 375 mg/m2, cycle 2-6: 500 mg/m2) therapy in relapse/refractory CLL patients. To test the targeting efficacy of MK2206 on Akt and downstream signals, we designed a one-week “run-in” of single agent MK2206 before the initiation of BR during cycle 1. MK2206 was then given along with BR on day 1 of cycles 2-6. Tumor response was evaluated 2 months after the 6th/last cycle of therapy based on IWCLL 2008 criteria. Correlative laboratory studies included assessment of Akt signaling and the plasma cytokine levels (multiplex beads assay (Invitrogen)) at baseline and after one week of first dose of single agent MK2206. ResultsNine patients (median age 68) were enrolled in the phase I portion of this trial. All 9 patients had unmutated immunoglobulin heavy chain variable region genes (IGHV) and 4 had del(11q). Six had received prior chemoimmunotherapy (CIT: FCR or PCR or PCO) and 5 had high risk disease defined as early progression within 26 months of the last CIT. Three had received alemtuzumab containing regimen. We have observed potent lymphocyte mobilization in all evaluated patients (n=7) after one-dose of MK2206 during week 1 (mean increase ALC: 35%) as well as reductions of plasma CCL3, CCL4 and CCL2 in the majority of the patients. A single dose-limiting toxicity (DLT) (febrile neutropenia and hemolytic anemia) was observed at dose level 1 (90 mg weekly) among 6 patients treated at this dose. Two patients with dose-limiting toxicities at dose level 2 (135 mg weekly) were observed where one patient had a rash and one patient had dehydration and febrile neutropenia among three patients treated at this dose. Accordingly, the 90 mg once weekly dose was determined to be the MTD for phase 2 trial testing. The most frequent grade 3 or 4 adverse events observed to date were neutropenia (44%) including febrile neutropenia (22%), rash (22%), diarrhea (22%), nausea and vomiting (11%), dehydration (11%), hemolytic anemia (11%) and thrombocytopenia (11%). The frequency of cytopenias was similar to the published German experience with BR (50% grade 3 or 4 hematological toxicities compared to 44% in N1087) in relapsed CLL patients (Fischer, JCO 2011). The median number of cycles received was 6 (range 1-6).Using an intent to treat analysis, two patients achieved complete response (CR, n= 2, 22%), one patient achieved nodular partial response (nPR, n=1, 11%) and five patients achieved a partial response (PR, n=5, 56%). The overall response rate (ORR) is 89%. ConclusionThe Akt inhibitor MK2206 in combination with BR is well tolerated in patients with relapsed/refractory CLL. Single dose of MK2206 inhibits Akt phosphorylation, mobilizes leukemic cells and decreases plasma cytokines involved in BCR signals. The preliminary efficacy of this combination demonstrated promising results with a 89% ORR and 22% CR albeit in the small phase 1 cohort. These results compare favorably to BR alone (9% CR and 59% ORR) in relapsed/refractory CLL patients. Phase 2 testing of this combination is now underway. AcknowledgmentThis work was supported by the funding from NCI grant K23CA160345 (WD), NCI grant CA95241 (NEK) and Alliance for Clinical Trials in Oncology (CA025224 and CA33601). Disclosures:Off Label Use: MK2206, used in a phase 1 trial of relapsed CLL disease. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Zent:Novartis: Research Funding; GlaxSmithKline: Research Funding; Biothera: Research Funding; Genzyme: Research Funding; Genentech : Research Funding. Reeder:Celgene: Research Funding; Novartis: Research Funding; Millenium: Research Funding.

Abstract CN04-04: Targeting death receptors and SMAC mimetics.

Abstract Evasion of apoptosis is a hallmark of human cancers and contributes to tumor formation and progression as well as resistance to current cancer therapies, which largely rely on intact cell death pathways in cancer cells. Apoptosis resistance can be caused by aberrant expression of antiapoptotic proteins, for example Inhibitor of Apoptosis (IAP) proteins, which are expressed at high levels in many human cancers. Since IAP proteins block apoptosis at the core of the apoptotic machinery by inhibiting effector caspases, they present promising therapeutic targets. Smac mimetics are small-molecule inhibitors that mimick the IAP-binding motif of the endogenous IAP antagonist Smac and are among the most widely used strategies to antagonize IAP proteins. In addition to their use as single agents, Smac mimetics have been used in combination with various cytotoxic stimuli to trigger cell death in cancer cells, in particular together with death receptor ligands such as soluble TRAIL or TRAIL receptor agonists. For example in pancreatic cancer_ one of the most lethal types of cancer_ IAP antagonists were shown to synergize with TRAIL receptor agonists to induce apoptosis and suppress tumor growth in preclinical in vitro and in vivo models. Furthermore, Smac mimetics and TRAIL acted in concert to trigger apoptosis in CLL, even in patients with poor prognostic features such chemoresistant disease, p53 mutation or chromosome 17p deletion. Thus, Smac mimetics may open new perspectives to overcome resistance to death receptor-mediated apoptosis in various forms of human cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN04-04. Citation Format: Simone Fulda. Targeting death receptors and SMAC mimetics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN04-04.

Dasatinib in combination with fludarabine in patients with refractory chronic lymphocytic leukemia: A multicenter phase 2 study

Resistance to chemotherapy-induced apoptosis in CLL is associated with overexpression of antiapoptotic proteins induced by signals from the microenvironment. In vitro, dasatinib effectively inhibits expression of anti-apoptotic regulators and restores fludarabine sensitivity in activated CLL. The aim of this study was to evaluate efficacy of one cycle of dasatinib monotherapy (100mg/day, days 1-28) followed by combination of dasatinib with fludarabine (40mg/m²/day, days 1-3 every 28 day) for a total of 6 cycles in fludarabine-refractory CLL. The primary endpoint was overall response rate according to the IWCLL'08 criteria. 20 patients were enrolled: 18 completed at least one cycle of treatment of which 67% finished at least 2 cycles of combination treatment. 3 of these 18 patients reached a formal PR (16.7%). Majority of patients obtained some reduction in lymph node (LN) size. Most frequent toxicity was related to myelosuppression. NF-κB RNA expression levels of circulating CLL cells decreased whereas the levels of pro-apoptotic NOXA increased during treatment. In conclusion, dasatinib/fludarabine combination has modest clinical efficacy in fludarabine-refractory patients.

NOTCH1 mutations influence survival in chronic lymphocytic leukemia patients

BackgroundNOTCH1 PEST domain mutations in chronic lymphocytic leukemia have recently been shown to be of prognostic relevance. Both NOTCH1 and NOTCH2 are constitutively activated in B-cell CLL but not expressed in normal B cells and may be involved in survival and resistance to apoptosis in CLL. We screened for mutations in different parts of both NOTCH1 and NOTCH2 genes and related the changes to survival and other known risk factors.MethodsIn a cohort of 209 CLL patients, we used single strand conformation analysis to determine which of the samples carrying the NOTCH mutations and direct dideoxy sequencing was used to determine the exact nucleotide changes. Kaplan-Meier curves and log rank test were used to determine overall survival for NOTCH1 mutated cases and Cox regression analysis was used to calculate hazardous ratios.ResultsIn the present study, we found NOTCH1 PEST domain mutations in 6.7% of the cases. A shorter overall survival was found in patients with NOTCH1 mutations compared to wildtype (p = 0.049). Further, we also examined the extracellular and the heterodimerisation domains of the NOTCH1 gene and the PEST domain and heterodimerisation domain of the NOTCH2 gene, but no mutations were found in these regions. NOTCH1 mutations were most commonly observed in patients with unmutated IGHV gene (10/14), and associated with a more aggressive disease course. In addition, NOTCH1 mutations were almost mutually exclusive with TP53 mutations. In the combined group of NOTCH1 (6.7%) or TP53 (6.2%) mutations, a significant difference in overall survival compared to the wildtype NOTCH1 and TP53 was found (p = 0.002).ConclusionsBoth NOTCH1 and TP53 mutations seem to be independent predictive markers for worse outcome in CLL-patients and this study emphasizes the contention that NOTCH1 mutations is a novel risk marker.

Abstract 3338: Novel caspase-3 activator, L14R8, induces apoptosis in chronic lymphocytic leukemia cells.

Abstract Chronic lymphocytic leukemia (CLL) is a B-cell malignancy and leukemia cells accumulate in bone marrow, lymph node, and peripheral blood due to decreased apoptosis. High levels of Bcl-2 family anti-apoptotic and inhibitors of apoptosis proteins (IAPs) in leukemic lymphocytes are primarily responsible for defective apoptosis in CLL. Both intrinsic caspase-9-mediated and extrinsic caspase-8-dependent apoptosis pathways activate downstream procaspase-3 to active casapce-3. In normal (n=4) as well as CLL lymphocytes (n=9), there is consistent expression of procaspase-3. However, active caspase-3 was present in normal lymphocytes but not in CLL cells suggesting that CLL cells have defective endogenous apoptosis. Hence, we hypothesized that compounds that can directly activate the functional ability of procaspase-3, should overcome the checkpoints of cell death and trigger apoptosis in CLL lymphocytes. Procaspase activating compounds (PAC; Vanquish Oncology) convert executioner procaspases to their active forms by chelating labile zinc on these enzymes. We have tested L14R8, a potent analogue of PAC, in primary CLL cells. The IC50 of L14R8-induced apoptosis in CLL was 7.7μM (n=12, 24hrs). L14R8 (10 μM) induced apoptosis as early as 6 hrs along with mitochondrial outer membrane permeabilization (MOMP). The drug significantly induced apoptosis in CLL patient samples (n=4) cultured in suspension cultures (60%, p=0.023,) as well as CLL lymphocytes under microenvironmental protection of marrow stromal cells (40%, p=0.011) albeit at a lower rate. Mechanistically, L14R8-treatment reduced uncleaved procaspases-3 and -9 expression and increased cleaved active caspases-3 and -9 expression (n=3). Consistent with the mechanism of action of L14R18 on terminal caspases, WT as well as cells lacking BAX and BAK (mouse embryo fibroblasts) were equally sensitive to this agent. Consistent with L14R8 mechanism, addition of exogenous Zinc ion completely reversed L14R8-mediated apoptosis of CLL lymphocytes (p=0.0063, n=4), but did not affect staurosporin-induced apoptosis in these lymphocytes. Moreover, L14R8-induced 86% (range 58-90%, n=4) cell death in CLL lymphocytes, which was reduced by pan caspase-inhibitors, Z-VAD to 58% (range 32-79%) and Q-VD-OPh to 35% (range 20-55%) cell death, which indicated the mechanistic recovery of L14R8-mediated apoptosis. Smac (the inhibitor of IAPs) protein expression was increased in CLL cells after treatment with L14R8. With respect to the therapeutic index, at 24 hrs, L14R8 induced 79% apoptosis in CLL cells (range 46%-97%, n=14) and only 18% (16-28%, n=3) in lymphocytes from healthy donors. We conclude that L14R8 induced apoptosis through activation of procaspase-3 to active caspase-3 in CLL cells. L14R8 and other PAC provide therapeutic index and may be developed into targeted therapeutics for patients with CLL. *Funded by Vanquish Oncology Citation Format: Viralkumar M. Patel, Kumudha Balakrishnan, William Wierda, Varsha Gandhi. Novel caspase-3 activator, L14R8, induces apoptosis in chronic lymphocytic leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3338. doi:10.1158/1538-7445.AM2013-3338

Differential Expression and Functions of NOTCH Family Members and Involvement of NR4A1 in the Regulation of Apoptosis in CLL

AbstractAbstract 3919Chronic lymphocytic leukemia (CLL) cells express constitutively activated NOTCH2 in a protein kinase C (PKC) dependent manner linking NOTCH2 to the activated state of the leukemic cells. The transcriptional activity of NOTCH2 is associated with the expression of CD23 and enhanced CLL cell viability. However, the regulation and possible functions of the individual NOTCH family members (NOTCH1–4) in CLL cells remain to be clarified.We took advantage of targeting nuclear NOTCH2 using the recently identified NOTCH2 transactivation inhibitor gliotoxin (WO 2006/135949). We also analysed the regulation and possible function of NOTCH1–4 in PKC stimulated CLL cells using a PMA model (Hubmann et al., BJH 2010) and a microenvironment model where CLL lymphocytes were co-cultured with primary bone marrow stromal cells (BMSC) (Shehata et al., BLOOD 2010).Electrophoretic mobility shift assays (EMSA) demonstrated that gliotoxin inhibited DNA-bound NOTCH2 complexes in PMA stimulated CLL cells in parallel to increasing the rate of apoptosis (mean±SD: 67±31% in gliotoxin treated cells versus 13±14% in the untreated controls, n=21). This was associated with downregulation of CD23A mRNA expression and CD23 surface expression (mean±SD: 42±32% versus 83±17%, n=21) as assessed by RT-PCR and FACS analysis. Exceptionally, one CLL case with a recently described NOTCH1 gain of function mutation appeared to be less sensitive to gliotoxin and had a persistent high expression of CD23.We next tested whether NOTCH2 inhibition by gliotoxin is a selective process or indirectly mediated by effects on proteasome regulated apoptosis. Proteasome assays showed that gliotoxin had a minimal or no effect on the chymotrypsin like activity of the proteasome in CLL cells. In addition, the activity of the proteasome regulated transcription factor NFκB and the expression of its target genes like BCL2 and MCL1 were also not influenced by gliotoxin. These data point to the selectivity of targeting NOTCH2 signaling by gliotoxin rather than indirectly through the regulation of proteasome activity.Short term (4 hours) exposure of CLL cells revealed that NOTCH1 was equally transcribed in unstimulated and in PMA activated CLL cells. NOTCH2 was upregulated in PMA activated CLL lymphocytes whereas NOTCH4 was only weakly detectable in unstimulated CLL cells. Gliotoxin treatment resulted in the downregulation of NOTCH1, NOTCH2 and NOTCH4 mRNA expression.Interestingly, the inhibition of NOTCH2 activity by gliotoxin was associated with the concomitant induction of NOTCH3 signaling especially in the presence of PMA. This was indicated by the induced mRNA expression of NOTCH3 and its preferred target gene HEY1. Moreover, the induced transcription of HEY1 correlated with the upregulation of NR4A1, a key regulator of apoptosis in activated lymphocytes. These data may thus point to a pro-apoptotic role for NOTCH3/HEY1/NR4A1 signaling in CLL cells. The data also suggest that gliotoxin induced apoptosis is associated with differential regulation of the anti-apoptotic and pro-apoptotic arms of NOTCH signalling in CLL cells.RT-PCR revealed that NOTCH1 and NOTCH2 are the main NOTCH family members which are expressed in CLL cells under co-culture conditions with BMSC and in freshly isolated CLL cells. Exposure to gliotoxin in co-culture selectively induced apoptosis in CLL cells and led to downregulation of NOTCH1 and NOTCH2 together with upregulation of NOTCH3 mRNA expression.In summary, the data suggest that nuclear NOTCH2 activity might protect activated CLL cells from apoptosis by modulating the expression of NR4A1. The induced expression of NOTCH3 and its target gene HEY1 by gliotoxin reveals the complex role of different NOTCH family members in the regulation of apoptosis in CLL cells. Therefore, the individual NOTCH receptors may have opposite effects on CLL cell viability which should be considered in therapeutic approaches aimed to target NOTCH signaling in CLL. Disclosures:No relevant conflicts of interest to declare.

Phenethyl Isothiocyanate (PEITC) Regulates Autophagy in Chronic Lymphocytic Leukemia.

AbstractAbstract 2906Chronic Lymphocytic leukemia (CLL) is currently incurable using conventional therapies. CLL cells can evade killing by various therapeutic strategies. However the precise mechanisms are currently unknown. Autophagy is regulated by a complex system of proteins, and is used by both normal and malignant cells as a protective mechanism against cellular stress induced by starvation, hypoxia, reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. In malignant cells autophagy was shown to promote tumorigenesis and/or resistance to chemotherapy. Therefore we hypothesized that autophagy may play a role in CLL biology.Autophagy can also promote cell death when stress signals are elevated above a particular threshold for a prolonged period of time. In this study we investigated the basal expression levels of autophagy specific genes and the effect of autophagy specific inhibitors (Bafilomycin, 3-methyladenine and hydroxychloroquine) and inducers (Phenethyl isothiocyanate) on CLL survival. Phenethyl isothiocyanate (PEITC) is about to enter clinical trials for CLL (NCT00968461).We have investigated induction of components of the autophagic pathway following treatment of CLL cells in vitro with a range of chemical inhibitors. Immunoblotting was carried out to investigate components of the autophagy pathway using phosphorylation state-specific and pan-reactive antibodies. Bafilomycin (BAF), 3-methyladenine (3-MA) and hydroxychloroquine (HCQ) toxicity towards CLL samples were evaluated by Annexin V/PI staining, MTT assay and immunoblotting for cleavage of the caspase 3 substrate poly(ADP ribose) polymerase (PARP) from its 116KDa to its 85KDa form. PEITC was used at concentrations between 2.5 and 25μM to investigate its effect on signaling. Autophagy was quantitated by immunoblotting of LC3-I and LC3-II. Lipidation of LC3 from LC3-I to LC3-II is a surrogate marker of autophagy and is essential for autophagasome formation. Immunoblotting was also performed for ATG3, ATG5 and ATG7, key components of the autophagy pathway. Monodansylcadaverine (MDC) was used with immunofluorescence and FACS analysis to investigate increases in autophagasome formation. Transmission electron microscopy (TEM) was used to confirm double membrane bound autophagosomes. Co-immunoprecipitation was used to evaluate if Beclin-1 was sequestered by Bcl-2 preventing autophagy. Its release from Bcl-2 enables Beclin-1 to interact with other autophagy specific proteins and initiates autophagasome formation.LC3-I was lipidated to LC3-II (p=0.019) and ATG3 (p=0.021) was upregulated to a greater extent in CLL samples compared with normal B-cell controls at basal levels. This suggested that autophagy was active to a greater extent in CLL samples compared with normal individuals. In addition Beclin was dissociated from Bcl-2 in CLL samples indicating that autophagy was active. Autophagy appears to be a pro-survival mechanism in untreated CLL cells as inhibiting basal levels of autophagy with autophagy inhibitors BAF (50–200nM), 3-MA (5–10mM) and hydroxychlorquine (5–10μM) resulted in CLL apoptosis as shown by MTT, Annexin V/PI analysis and PARP cleavage.Interestingly augmenting autophagy was also capable of inducing apoptosis in CLL samples. Treatment with PEITC caused an increase in punctate staining using MDC which is suggestive of autophagosome formation. We went on to determine that PEITC further induced LC3-II lipidation using immunoblotting and showed a substantial increase in overall LC3 protein expression. PEITC also induced the expression of ATG3, a key protein in the autophagy pathway. We then evaluated autophagosome formation using TEM (Figure 1). Our data showed greater numbers of autophagosomes in the PEITC treated samples compared to the untreated controls.Therefore autophagy in CLL sits on a knife-edge, such that perturbations that either increase pro- death or decrease pro-survival autophagy signals can result in CLL cell death, depending on the duration and intensity of the signal. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Results of the Phase 1 Trial of RG7112, a Small-Molecule MDM2 Antagonist, in Acute Leukemia

AbstractAbstract 675 Background:Activation of the p53 pathway by inhibition of its negative regulator MDM2 has been proposed as a novel strategy for cancer therapy. We report the final results of the Phase 1 leukemia trial of RG7112, a small molecule antagonist of MDM2 which activates p53 by disrupting the p53-MDM2 interaction. Methods:The trial was designed as a multi-center, open-label Phase 1 MAD study of patients with relapsed/refractory leukemia treated with escalating doses of single agent RG7112 in two strata: A:AML, ALL, CML-BC; and B:CLL and S-CLL. RG7112 was administered orally daily for 10 days followed by 18 days of rest. Primary objectives were to determine MTD and DLT; secondary objectives to assess PK, PD and clinical responses. Enrollment is complete. 4 patients remain on study, and final data is being compiled. Peripheral blood was collected for PK and biomarkers pre-treatment, at multiple time points during the first 48 hours, and on d10. Bone marrow was collected pre-treatment, d10 and d28. Blood and marrow were purified by MACS sorting (CD19, 33 or 34 Ab). AmpliChip analysis of p53 mutational status (single nucleotide alterations exons 2–11) was performed. MIC-1 (macrophage inhibitory cytokine-1), a p53 transcriptional target, was analyzed in serum by ELISA as a pharmacodynamic marker of p53 activation. Baseline MDM2 mRNA levels, and changes from baseline, as a marker of p53 activation and feedback on MDM2, were measured by RT-PCR. A panel of 24 genes was assessed serially by TLDA (q-PCR). Results:116 patients (84 AML,10 ALL, 2 CML, 20 CLL/sCLL) were treated with RG7112 doses ranging from 20 – 2430 (1215 BID) mg/m2/day × 10 days. The dose-exposure relationship (Cmax, AUC, and Ctrough) was approximately linear with a median half-life of 1–1.5 days. 43 patients with acute leukemia were dosed at the MTD of 1500 mg BID (flat dose) × 10 days. Major adverse events(> 20%) were nausea, diarrhea and vomiting. Other major (>10%) AEs were complications of leukemia (e.g. neutropenic fever). Clinical activity was noted during dose escalation (ASH 2011), including apoptosis in AML and CLL, CR in an AML patient (360mg/m2) stable for 6 cycles (10 months) and one patient with CLL/Richter’s transformation with stable disease (40 mg/m2) for >25 cycles. New data focuses on AML patients treated at MTD. This cohort received a median of 3 prior treatments, 16 were refractory to last treatment. 17 (40%) had evidence of hematologic improvement (greater than 50% drop in peripheral and/or BM blasts by d28). Of the 31 patients who completed cycle 1 dosing, 5 patients (16%) had a CR (2 CR, 2 CRi. 1 CR without recovery). Two CR patients have proceeded to allotransplant. All CR patients’ blasts were p53wt. Of the 12 additional patients with single-agent hematologic activity, 3 had biochemical evidence of tumor lysis syndrome, 10 had p53status evaluable, and mutations were detected in 3: (exon 7 missense, exon 9 frameshift, intron 7 splice).p53 activation by RG7112 was demonstrated by MIC-1 induction in serum. MIC-1 levels were elevated at RG7112 plasma concentrations >2 μg/mL or AUC24h >50 μg · h/mL and were highly correlated (0.5629) with exposure to RG7112 (AUC24h) at steady state following a sigmoidal Emax model. Preliminary data review suggests a positive correlation between exposure to RG7112 and changes in MDM2 gene expression at day 2. In addition, q-PCR analysis demonstrated exposure-dependent induction of key p53 transcriptional targets including MDM2, p21, and PUMA. p53 protein increase by WB was demonstrated at the MTD in selected patients. These findings are consistent with the proposed mechanism of action of RG7112. Conclusions:We report evidence of single agent clinical activity in patients with leukemia treated with RG7112, including 6 AML patients with CR, 5 of 31 (16%) at the MTD, and additional patients with significant decreases in blasts. Concentration-related pharmacodynamic biomarker activity of the p53 pathway was demonstrated by increases in MIC-1 levels, and induction of p53 target genes including mdm2, p21 and PUMA. 4 patients remain on treatment and final results will be reported. We provide proof of mechanism and evidence for single-agent clinical activity of MDM2 antagonism in leukemia by demonstrating complete remission, lysis of leukemic blasts and activation of p53 pathway targets in leukemic cells from patients treated with RG7112. Clinical studies of RG7112 in combination are underway. Disclosures:Andreeff:Hoffmann-La Roche: Research Funding; Karyopharm Therapeutics: Unrestricted gift, Unrestricted gift Other. Yee:Roche: Membership on an entity's Board of Directors or advisory committees. Assouline:Roche: travel funding Other. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy; Ariad: Consultancy. Drummond:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Geho:Roche: Employment. Blotner:Roche: Employment. Cheng:Roche: Employment. Vassilev:Roche: Employment. Ding:Roche: Employment. Zhi:Roche: Employment. Middleton:Roche: Employment. Nichols:Roche: Employment.

The p90RSK Inhibitor BI-D1870 Induces Apoptosis in CLL Cells

Abstract 2492Chronic Lymphocytic leukaemia (CLL) is the most common leukemia in the western world but is currently incurable using conventional therapies. Therefore novel agents which can kill CLL cells either alone or in combination with conventional agents are being continually sought. Activation of the MAPK pathway has been implicated as one of the protective signaling pathway in CLL and is characterised by expression of phosphorylated ERK. In CLL cells ERK can be activated by phosphorylation after BCR activation with αIgM. However in a proportion of CLL isolates ERK is constitutively active. MEK inhibitors (MEKi) are currently being used in clinical trials to treat relapsed or refractory leukemia including CLL (www.clinicaltrials.gov, NCT00920140). Therefore we hypothesised that inhibiting the ribosomal S6 kinase p90RSK which is immediately downstream of the MAPK pathway, may be of therapeutic value for the treatment of CLL. Since p90RSK regulates the majority of the anti-apoptotic signaling of the ERK pathway, we investigated the effect of inhibiting p90RSK with its specific inhibitor BI-D1870 (BI-D) on apoptosis in CLL.Immunoblotting was carried out to investigate phosphorylation of components of the ERK/p90RSK pathway using phosphorylation state-specific and pan-reactive antibodies. BI-D toxicity was evaluated by Annexin V/PI staining, MTT assay and immunoblotting for cleavage of the caspase 3 substrate poly(ADP ribose) polymerase (PARP) from its 116KDa to its 85KDa form.BI-D treatment of CLL cells decreased S6 phosphorylation, and this down regulation correlated with apoptosis. BI-D killed CLL cells rapidly, with a mean IC50 of 12μM following 18h incubation. Treatment of CLL cells with BI-D induced cytochrome C release indicating inhibition of p90RSK induced apoptosis by the intrinsic death pathway. However its toxicity was not due to inhibition of protein translation since inhibition of mTOR, a parallel pathway which also regulates S6 phosphorylation, was minimally toxic to CLL cells at concentrations as high as 500nM. However apoptosis did coincide with a reduction of the antiapoptotic protein Mcl-1. We further demonstrated that BI-D augmented apoptosis induction by cytotoxic drugs, as shown by PARP cleavage and by annexin V/PI binding assays. Analysis of cytotoxicity data using CalcuSyn software provided evidence for synergistic killing of the majority of CLL isolates by combinations of BI-D with either chlorambucil, fludarabine or nutlin 3.Constitutive or antigen induced activation of p90RSK via the MAPK pathway in CLL cells may contribute to apoptosis resistance and its inhibition may be of value in devising novel therapeutic strategies, either alone or in conjunction with conventional cytotoxic drugs. p90RSK inhibition led to apoptosis independently of protein translation inhibition and may be attributable in part to downregulation of Mcl-1. Given the recent interest in using MEK inhibitors (MEKi) for the treatment of CLL, these data suggests MEKi may induce apoptosis in CLL in part by inhibiting pathways downstream of p90RSK. In addition BI-D is a highly specific inhibitor of p90RSK and therefore may negate the off target effects shown with some MEKi. This is the first report on the consequences of inhibition of p90RSK in CLL cells and suggests that inhibiting p90RSK may be of considerable interest for the treatment of CLL. Disclosures:No relevant conflicts of interest to declare.

TRIS (DIBENZYLIDENEACETONE) Dipalladium a Small-Molecule Palladium Complex Is Effective in the Induction of Apoptosis for B-Chronic Lymphocytic Leukemia B-Cells

Abstract 2851 Background:In an attempt to better treat B-Chronic Lymphocytic Leukemia (CLL) patients, we have examined a unique organometallic complex called Tris (dibenzylideneacetone) dipalladium (Tris DBA). This novel agent is a small-molecule palladium complex shown to have antiproliferative activity against melanoma cells and possible antitumor activity. Tris DBA is known to inhibit activation of signaling molecules such as PI3K and STAT-3, critical factors in CLL B-cell survival and resistance to therapeutic agents. Because B-CLL remains incurable and treatment strategies challenging, our findings may be useful in pursing new therapies to improve patient care. Therefore, this information prompted us to investigate whether Tris DBA could induce apoptosis in CLL B-cells.Method of Approach: Primary leukemic B-cells from CLL patients were cultured in serum free media and treated at 2 × 106 cells per ml with vehicle or various doses of Tris DBA (1 – 10 μg/ml) for 24 – 72 hours. After treatment, cells were analyzed for apoptosis by a FITC-labeled annexin-V/propidium iodide assay using the FACScan. To assess changes in anti-apoptotic proteins, CLL-B cells were cultured for 24 hours with either vehicle or 2.5μg/mL of Tris DBA. Total protein extracts were used for immunoblot methodology with specific antibodies. In some experiments we developed primary marrow stromal cells (MSC) that when nearly confluent were used for co-culture experiments in the presence of freshly isolated CLL B-cells with various chemotherapeutic drugs and/or Tris DBA. Finally, we compared CLL patients with known immunoglobulin heavy chain variable region (IgVH) status because of the clear differences in clinical outcome for these two prognostic subgroups. Results:CLL B-cells were shown to be sensitive to Tris-DBA with a steep dose response curve that significantly increased apoptosis over control levels as early as 24 hours of culture. Thus the LD50 of Tris DBA was between 2.5 and 5.0 μg/ml for the CLL B-cells who were IgVH mutated while between 5 and 7.5μg/ml for the unmutated status CLL B cells (n=10). Normal B or T lymphocytes were found to be equally sensitive to Tris DBA when compared to CLL B-cells however mesenchymal stromal cells (MSC) from CLL marrow (CLL MSC) were far less sensitive to the Tris DBA drug. Thus the LD50 for normal T cells, B cells or NK cells was between 2.5 and 5.0 μg/ml while the LD50 for the MSC was not reached over the dose range of 1–10 μg/ml. When CLL B-cells were added to CLL derived primary MSC in the presence of increasing concentrations of Tris DBA (2.5–10 μg/ml), the cytotoxic impact of Tris DBA on CLL B-cells could be overcome in the presence of MSC. To delineate the mechanism of apoptosis induction of CLL B-cells, we treated CLL B-cells with sub-lethal dose of Tris DBA and prepared cell lysates for Western blot analyses. We found that the anti-apoptotic proteins XIAP and Bcl-2, but not Mcl-1, were down regulated in Tris- DBA-treated leukemic B-cells. In addition, phosphorylation levels of Akt and STAT-3 were inhibited with exposure to Tris DBA as measured by Western blot using specific antibodies. Finally, we studied if Tris DBA could be synergistic or additive with fludarabine in induction of apoptosis of CLL B- cells. Interestingly, we found that in 2 of 3 assays there was synergy between Tris DBA and fludarabine in the enhancement of CLL B-cell apoptosis. Conclusions:We have established that a unique compound, Tris dipalladium, is effective at inducing apoptosis in CLL B-cells whether these leukemic cells are IGVH mutated (low risk) or unmutated (high risk). In addition this compound can modify the levels of XIAP and Bcl-2, Akt and STAT3 activation in the leukemic B-cells. Importantly, by itself the Tris DBA is effective even in the presence of stromal cells albeit losing some effectiveness at high doses in the co-culture and can generate synergy when added to a purine nucleoside for induction of B-cell apoptosis. In total these findings support future clinical applications of this compound in treating CLL patients. Disclosures:Kay:Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity’s Board of Directors or advisory committees. Arbiser:Natuderm: Patents & Royalties.

Abstract 2138: Vinblastine acutely sensitizes hematopoietic cancers to Bcl-2 targeting drugs

Abstract Chronic lymphocytic leukemia (CLL) characteristically maintains high levels of the anti-apoptotic protein Bcl-2, thus protecting them from apoptotic death and leading to an accumulation of mature resting B cells. Many other hematopoietic malignancies including acute promyelocytic leukemia (APL) also depend upon Bcl-2 family proteins for survival, suggesting that Bcl-2 proteins are excellent targets for anti-cancer therapies. We have previously shown that some leukemia and lymphoma cell lines undergo rapid apoptosis (≤6 h) when incubated with the microtubule disrupting agent vinblastine. Several other cell lines undergo this rapid apoptosis when vinblastine is combined with various approaches that target the anti-apoptotic proteins Bcl-2 or Mcl-1. These approaches include incubation with ABT-737 that inhibits Bcl-2 and Bcl-X, and SCH727965 (dinaciclib) that inhibits CDK7/9, thereby inhibiting translation which results in rapid loss of Mcl-1. In this study, we have assessed the ability of these agents alone or in combination to induce apoptosis in freshly isolated CLL and APL cells. Incubation with vinblastine alone induced variable apoptosis within 6 h (5-60%). ABT-737 induced rapid and almost complete apoptosis in CLL and in 50% of APL cells ex vivo. However, when combined with vinblastine, a 10-fold lower concentration of ABT-737 was required to induce complete apoptosis. Dinaciclib alone also induced variable levels of apoptosis (5-90%), but again the combination with vinblastine induced extensive apoptosis at lower concentrations of both drugs. Normal peripheral blood mononuclear cells were resistant to single agent or combination treatments. Sensitivity to ABT-737, dinaciclib or vinblastine was independent of CD38 and ZAP70 status and previous clinical treatment (untreated or relapsed patients). These results suggest that combinations of vinblastine and ABT-737 or dinaciclib are more effective than single agent therapy. Furthermore, these combinations may overcome resistance to individual drug therapies in patients with CLL or APL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2138. doi:10.1158/1538-7445.AM2011-2138

Apoptosis induces Bcl-X S and cleaved Bcl-X L in chronic lymphocytic leukaemia

The Bcl-X gene has both pro-survival, Bcl-X L, and pro-apoptotic, Bcl-X S, gene products, which are produced by alternative splicing. The function of these proteins has previously been characterised in cell lines, often by transfecting expression constructs, and primary cell systems capable of dynamically regulating Bcl-X L and Bcl-X S have not been described. Such a system is potentially important to allow testing of agents that promote apoptosis by increasing the amount of Bcl-X S at the expense of Bcl-X L. In this report we characterise Bcl-X gene products in primary human leukaemic B-cells in culture conditions associated with survival and apoptosis. We found that Bcl-X S was induced in spontaneous and drug-induced apoptosis and that apoptosis induced in cells cultured on mouse fibroblasts expressing CD40 ligand with IL-4 (CD154/IL-4), a condition mimicking the tissue microenvironment, additionally produced expression of cleavage products of Bcl-X L. Both Bcl-X S and Bcl-X L were produced in a caspase dependent manner. We tested emetine, an agent previously reported to increase Bcl-X S but found that it did not have this effect in primary human B-cells. Therefore, there are two mechanisms—cleavage of Bcl-X L and production of Bcl-X S—by which Bcl-X gene products could enhance apoptosis in CLL but neither appeared to have a primary role in inducing leukaemic cell death.

Targeting Destruction of Mcl-1 by Activation of Protein Phosphatase 2A In CLL and B-Cell Lymphomas

AbstractAbstract 4190 Background and Significance:Myeloid Cell Leukemia-1 (Mcl-1) is an anti-apoptotic protein that is elevated in fludarabine-resistant CLL patients. Expression levels of Mcl-1 correlate with time to first treatment and overall survival in CLL patients. Furthermore, elevated levels of Mcl-1 have also been reported in B-cell lymphomas. The regulation of Mcl-1 stability occurs through phosphorylation of several serine and threonine residues catalyzed by constitutively activated kinases. The tumor suppressor protein phosphatase 2A (PP2A) is important in deactivation of several kinases: Akt, the mitogen activated protein kinases (MAPK) p38, JNK, ERK, and NFkB (through IkK). We have discovered novel peptides that antagonize the SET oncoprotein, a potent physiological inhibitor of PP2A, and increase cellular PP2A activity. Importantly, we note that SET is overexpressed in CLL and B-cell lymphoma cells and that treatment of these cells with SET antagonist peptides is cytotoxic for the malignant cells both in vitro and in vivo. We show here that activation of PP2A by antagonism of SET results in reduced Mcl-1 levels and induction of apoptosis in CLL and B-cell lymphoma cells. Methods:Patients were from the Duke University and V.A. Medical Centers, and normal controls were from the community. Blood CLL cells and control normal B-cells were purified using negative selection with antibodies. The human Ramos and Raji B-cell lymphoma cell lines were from ATCC. We determined cytotoxicity using the MTS colorimetric assay. SET antagonist peptides were prepared by chemical synthesis. Western blotting and immunoprecipitation were performed with antibodies to SET, Mcl-1, PP2Ac, c-Myc, Axin, Pin-1, GSK3beta, Bcl-2, Bcl-XL and beta-actin. Apoptosis assays were performed with the annexin-V/propidium iodide staining method. Results:We previously demonstrated SET overexpression in CLL cells and that SET antagonist peptides activate PP2A and are cytotoxic for malignant B-cells. Using freshly-isolated CLL cells and Raji and Ramos cell lines, cytotoxicity of the SET antagonist peptide COG449 was evaluated. We found the concentrations for 50% cytotoxicity (ED50) to be 77 nM for CLL cells, 125 nM for Ramos cells, 250 nM for Raji cells, and >10,000 nM for normal B-cells. Annexin-V staining indicated that apoptosis was induced at concentrations comparable to the ED50s for cytotoxicity of the compounds tested. COG449 treatment of NOD/SCID mice bearing tumors after xenografting with Ramos cells resulted in reduced tumor growth. After treatment for 8 days, there was a 61% reduction in final tumor mass at harvest on day 19 (p<0.001). To evaluate the induction of apoptosis, we treated CLL or Raji cells with COG449 and monitored the cellular levels of the anti-apoptotic Bcl-2, Bcl-XL, and Mcl-1 proteins. We found no significant changes in Bcl-2 or Bcl-XL levels, but Mcl-1 levels decreased in a dose dependent manner. Immunoprecipitation of Mcl-1 was performed to identify proteins that interact with Mcl-1 and regulate its stability. We identified a novel pattern of immunoprecipitating proteins and found that PP2A and SET are part of the Mcl-1 regulatory complex. Conclusions:The observation that SET antagonist peptides induce apoptosis through dose-dependent reduction in the cellular level of Mcl-1 indicates that PP2A may modulate the stability of Mcl-1. Co-immunoprecipitation of SET and PP2A with Mcl-1 suggests that a novel complex exists for the regulation of Mcl-1 stability and that PP2A-mediated dephosphorylation of Mcl-1 may be a critical step in this process. Induction of Mcl-1 degradation with PP2A reactivation therapy may be a useful approach for the treatment of CLL and other B-cell malignancies. Disclosures:Christensen:Cognosci: Employment, Equity Ownership. Oddo:Cognosci Inc.: Employment. Vitek:Cognosci Inc.: Employment, Equity Ownership.

GlycoVariant Anti-CD37 Small Modular Immuno-Pharmaceutical Exhibits Superior Natural Killer Cell Mediated Cytotoxicity Against Chronic Lymphocytic Leukemia Cells at Low Concentrations and Low Antigen Density

AbstractAbstract 1847CD37 is a tetraspanin transmembrane family protein that is strongly expressed on the surface of mature human B cells and transformed mature B cell lymphoma and leukemia cells, including CLL cells. It is absent or minimally expressed on normal T cells, NK cells, monocytes, and granulocytes. Predominant expression of CD37 on CLL cells makes it an ideal candidate to target with potential agents for treatment of CLL. TRU-016, a small modular immunopharmaceutical protein (SMIP) that specifically binds to an extracellular region of CD37, is presently in clinical trials in CLL patients. TRU-016 includes humanized immunoglobulin variable regions (scFv) fused to a human IgG1 Fc region. We have previously reported that SMIP-016, the chimeric version of the humanized TRU-016, induced apoptosis in CLL B cells in the presence of goat anti-human Fc antibody cross-linker through a novel, caspase-independent pathway. Furthermore, SMIP-016 showed potent in vivo activity in a SCID xenograft mouse model. Aside from direct cytotoxicity, SMIP-016 mediates antibody-dependent cellular cytotoxicity (ADCC) by NK cells both in vitro and in vivo. In an attempt to enhance its ADCC function, a new variant of SMIP-016, SMIP-016GV, was designed with a modification of the glycosylation of the Fc portion of the molecule. SMIP-016GV exhibits enhanced binding to both low- and high-affinity molecular variants of human CD16 (FcγRIII) and augments ADCC potency when compared to SMIP-016. In this study, we compared SMIP-016GV and SMIP-016 in direct cytotoxicity and ADCC against CLL B cells. While SMIP-016 and SMIP-016GV mediated comparable direct cytotoxicity at 48 hrs in the presence of goat anti-human Fc crosslinker, the SMIP-016GV resulted in 2 to 4 fold increase in NK cell mediated ADCC function at all effector to target ratios tested. This increased ADCC with SMIP-016GV was observed using NK cell effectors derived from both normal as well as CLL-affected individuals. In addition, this enhanced cytotoxicity was sustained at concentrations of SMIP-016GV as low at 5E-6 μg/ml. These low concentrations of SMIP-016GV were also able to mediate superior ADCC in 697 cells expressing as few as 10,000 molecules of surface CD37 antigen. Furthermore, NK cells stimulated with the glycovariant were potently activated and released 3 to 4 fold more IFNγ compared to SMIP-016. Ongoing studies are aimed at defining other effector cells which may interact with SMIP-016GV via different Fcγ Receptors. Collectively, these results suggest potential use of the SMIP-016GV with enhanced ADCC function as an alternate for TRU-016 in B cell malignancies including CLL therapy. Disclosures:Siadak:Trubion Pharmaceuticals: Employment.