Strege, Ann K.

Abstract Background : B-Chronic Lymphocytic leukemia (CLL) is the most common leukemia in North America. The standard current treatments use purine nucleoside analogues as single agent therapy or in combination with rituximab, steroids, and alkylating agents. Overall response rates with these treatments in previously untreated patients may reach 90 % with CR rates of 50–60%, however, most patients will relapse. Thus, despite refinements in therapy, CLL remains an incurable malignancy and there remains significant and urgent need to identify and develop new agents with novel mechanisms of action for the treatment of CLL. In line with this we have explored the mechanism and in vitro activity for a tyrphostin, adaphostin. Methods: We evaluated the in vitro efficacy of adaphostin to induce apoptosis in CLL B-cells. Peripheral blood was collected from patients with CLL (n=57). Highly purified CLL B-cells ((minimum 80% CD19+, mean 92.2% CD19+ and 94.4% CD19+/CD5+ positive)) were cultured with freshly prepared adaphostin for 24 – 120 hours. Cell death was analyzed by flow cytometry using Anexin V/Propidium iodide (PI). PARP cleavage and anti-apoptotic protein levels were measured by immunoblot techniques. The effect of combination treatment on CLL B cells with adaphostin and fludarabine on CLL B-cells was also assessed. Results: Analysis by Annexin V/PI staining revealed that the mean IC50 for adaphostin at 24 h was 4.2 uM (range 1.10 uM-11.25 uM; median = 4.25; n = 29) for CLL isolates and >10 uM for B and T-cells isolated from normal donors. Median IC50 levels for Adaphostin were not significantly different based on IgVH mutation status, level of CD38 expression, or cytogenetic abnormalities by FISH testing (Table 1). Immunoblots demonstrated adaphostin-induced PARP cleavage and cleavage of caspase 3 substrates, suggesting that adaphostin induces cell death through apoptosis. Adaphostin increased the intra-cellular level of reactive oxygen species (ROS) in CLL B-cells; and the antioxidant N-acetylcysteine blocked both adaphostin-induced ROS generation and apoptosis (mean reduction cell death=60%; range 23–99% reduction) suggesting generation of ROS is critical to adaphostin’s induction of apoptosis. Adaphostin also caused a decrease in the level of the anti-apoptotic proteins Bcl-2 in a majority of patients on both flow cytometry and immunoblots. When adaphostin was combined with fludarabine (F-ARA-ATP), a synergistic effect on cell death was observed in all 10 CLL samples when analyzed by mathematical modeling software. Summary: These findings indicate that adaphostin induces selective apoptosis in CLL B-cells from all risk categories through a mechanism that involves ROS generation. Importantly, we also demonstrate its ability to augment the effects of fludarabine. We continue to explore the preclinical development of adaphostin as a novel agent for the treatment of CLL. IC50 Adaphostin Dose Levels by Prognostic Groups FACTOR N Median IC 50 Adaphostin Range p-Value Rai Stage Group Low/Int (0–II) 19 4.7 1.8 – 11.25 0.018 High (III – IV) 10 3.05 1.1 – 5.4 - IgVH Mutation Status Mutated 11 4.45 1.1 – 11.25 0.72 Nonmutated 10 3.93 1.8 – 5.8 - CD38 Status Negative 20 4.55 1.1 – 11.25 0.74 Positive 9 4.25 1.8 – 6.6 - FISH defects 13q- 8 4.70 1.75 – 11.25 Normal 6 3.53 1.1 – 5.8 0.83 12+ 4 3.93 2.25 – 6.6 17p/11q 5 4.20 2.0 – 5.4

Abstract We have found that CLL B cells spontaneously secrete vascular endothelial growth factor (VEGF) and that a VEGF autocrine pathway can induce apoptosis resistance in these cells. Recently, we also found that hypoxia-inducible factor-1 alpha (HIF-1α) is highly expressed in CLL B cells. Since this protein is a potent transcription factor for the induction of VEGF, we were interested in further definition of HIF-1α regulation and its function in CLL B cells. CLL blood B cells overexpress HIF-1α protein but not mRNA for HIF-1α compared to normal blood and splenic B cells. Immunohistochemistry (IHC) showed that circulating blood CLL B cells and a subset of CLL marrow cells uniformly express HIF. Hypoxic conditions (i.e., 1% O2) did not increase the protein levels of HIF-1α nor mRNA for HIF-1α in CLL B cells, indicating that the high HIF-1α protein level is due to post-translation modification. Blockade of signaling pathways known to increase HIF-1α levels also did not alter the high levels of HIF-1α in CLL B cells. IHC and nuclear extraction assay demonstrated that HIF-1α was predominantly located in the CLL B cell nucleus. In addition, the nuclear extract when immunoprecipitated for HIF-1α was shown to be complexed with the co-activator p300, indicating that HIF-1α is transcriptionally active. Co-immunoprecipitation assay showed that HIF-1α from CLL B cells does not associate and form a complex with von Hippel-Landau protein tumor suppressor (pVHL), indicating that the proteasome dependent degradation pathway for HIF-1α protein in CLL B cells is dysfunctional. Using immunoblot or IHC methods, we were unable to detect pVHL protein in CLL B cells; however, we were able to use immunoprecipitation of CLL B cell lysates to demonstrate there is pVHL in CLL B cells. Prolyl hydroxylases (PHD 1, 2, and 3) are negative regulators for HIF-1α via hydroxylation of amino acid prolines in the oxygen degradation domain (ODD) which permits interaction with pVHL. RT-PCR results revealed that there is a subset of CLL patients who had ≥ 50% reduction of PHD 1 and 3 mRNA levels. However using a hydroxylation specific polyclonal antibody we found that HIF-1α from CLL B cells is indeed hydroxylated. Finally, silencing of HIF-1α by RNA interference in CLL B cells was associated with a selective decrease in VEGF mRNA levels but not VEGF-R1, Mcl-1 and prolyl hydroxylases (PHD 1–3) other downstream target genes of HIF-1α. These data show that the high endogenous HIF-1α levels in CLL B cells are due to a defect in HIF-1α degradation via the proteosomal pathway. We believe that this abnormality is linked to the autocrine VEGF pathway in CLL B cells and ultimately results in increases in their apoptotic resistance. Inhibition of HIF-1α levels may be of therapeutic benefit to CLL patients.

Abstract CLL B cells have significant opportunity to interact with multiple tissue sites that are critical to the leukemic cell’s ability to survive. The marrow stromal elements (MSE) in B-chronic lymphocytic leukemia (B-CLL) are very likely one of the most frequently involved in this important cell-cell interaction. We have utilized a novel bone marrow biopsy technique (Alvi S, Leuk Res, 2001) that consistently generates robust and long-lived marrow stromal elements from B-CLL patients in order to study CLL B cell apoptosis status in relation to angiogenic factors. The latter angiogenic parameters were studied as we have previously detected both a VEGF based autocrine pathway for CLL B cells and found an extensive neovascularization in CLL marrows. Bone biopsy material from B-CLL patients (N=25) yielded active cell growth in vitro which expresses the features of marrow stromal constituents. Importantly, these marrow constituents were able to be sustained for several months (range, 3 – 52 weeks) and also could be transferred repetitively to new culture dishes. CLL B cells were able to spontaneously release VEGF and TSP-1 as we have previously published (Kay N, Leukemia, 2002). MSE from CLL marrow (N=5) were found to spontaneously secrete basic fibroblast growth factor (bFGF) 5.2 ± 0.9 pg/ml (mean ± one SEM), vascular endothelial growth factor (VEGF) 162 ± 48 pg/ml and thrombospondin-1 (TSP-1) 90 ± 26 ng/ml into the culture medium. Significant reduction in CLL B cell spontaneous apoptosis,measured by Annexin/PI incorporation (mean ± one SEM), was seen after exposure to the CLL MSE (N=6) both after 24 hours (i.e., Non exposed CLL B cells vs. MSE co-cultured CLL B cells was 70% ± 9 vs. 39% ± 10 respectively for 24 hours, p < 0.01) and 72 hrs (data not shown). Exposure to MSE also enhanced the in vitro drug resistance of CLL B cells to chlorambucil (C). Thus, for CLL B cells (n=5) cultured with CLL MSE and C (1μM) for 24 hours, apoptosis levels were reduced from 58.8% ± 7.8 for B cells without MSE compared to 39.8% ± 3.8 for B cells exposed to MSE. Apoptosis resistance enhancement for CLL B cells was found to be similar with normal marrow stromal elements and a stromal cell line (HS-5). Anti-apoptotic proteins detected by immunoblot were found to increase for CLL B cells exposed to stromal elements from bone marrow biopsies and included Mcl-1, Bcl-2, Survivin and XIAP. While Mcl-1 was found to increase, there was a decrease in Bcl-2 and Survivin detected by immunoblot when CLL B cells were exposed to the HS-5 cell line implying a different mechanism for apoptosis enhancement. When CLL B cells were added to CLL MSE for 24 hours, there was an increase in VEGF released into the culture medium (283 pg ± 114 for the co-culture vs. 14 pg ± 1.7 and 162 pg ± 48.8 for CLL B cells and MSE cultured alone respectively). In contrast co-culture of CLL B cells with MSE (24 hours) resulted in less TSP-1 in the culture medium than expected (106 ng ± 28.8 for co-cultured CLL and MSE cells vs. 89.5 ng ± 27.5 and 90.9 ng ± 26.4 for CLL B cells and MSE cultured alone respectively). This change in the ratio of pro- to anti-angiogenic factors favoring VEGF strongly suggests that CLL B cell interaction with stroma can facilitate an angiogenic switch and may be linked to disease progression. In addition, we have found that bone biopsies from B-CLL patients can generate long-term marrow stromal elements that are able to reliably enhance CLL B cell apoptosis resistance.

AbstractObjectivesTo analyze the economic impact of testing for activated protein C resistance (APC-R) due to factor V Leiden (FVL) mutation with APC-R with reflexive FVL genotyping (algorithmic approach) or genotyping alone.MethodsOptumLabs Data Warehouse (OLDW) data were used to assess testing approaches. Insurance claims for APC-R and FVL in 2013 were compared with the Mayo Clinic database. Centers for Medicare & Medicaid Services diagnostic fee schedules were used to assign costs.ResultsOf 19.3 million OLDW-covered individuals, 74,242 (0.385%) received 75,608 tests: APC-R, 2,265 (2.9%); FVL genotyping, 70,619 (90.1%); and both APC-R and FVL, 2,724 (7.0%). In total, 1,317 tests were performed at Mayo Clinic: APC-R with reflex FVL (1,256; 95.4%) and FVL alone (61; 4.6%). Costs per evaluated individual and per total population (person/year) in OLDW and algorithmic approach were $83.77 vs $36.38 and $0.32 vs $0.14, respectively.ConclusionsThe cost-optimized algorithmic approach reduces health care costs.

AbstractB-cell chronic lymphocytic leukemia (CLL) is characterized by accumulation of clonal lymphocytes resistant to apoptosis. We evaluated the ability of the investigational antileukemic agent adaphostin to induce apoptosis in CLL B cells and synergize with fludarabine in vitro. Analysis by annexin V/propidium iodide (PI) staining revealed that the concentration of adaphostin required to induce 50% cell death (IC50) at 24 hours was 4.2 μM (range, 1.10-11.25 μM; median, 4.25 μM; n = 29) for CLL isolates and more than 10 μM for B and T cells from healthy donors. Immunoblots demonstrated adaphostin induced poly(adenosine diphosphate-ribose) polymerase (PARP) cleavage and cleavage of caspase-3 substrates, suggesting that adaphostin induces apoptosis. Adaphostin increased the level of reactive oxygen species (ROS) within CLL B cells, and the antioxidant N-acetylcysteine blocked both adaphostin-induced ROS generation and apoptosis. Adaphostin also caused a decrease in the level of the antiapoptotic protein Bcl-2. When adaphostin was combined with fludarabine (F-ARA-AMP), a synergistic effect on cell death was observed in all 10 CLL samples. These findings not only indicate that adaphostin induces apoptosis selectively in CLL B cells through a mechanism that involves ROS generation but also demonstrate its ability to augment the effects of fludarabine. Further preclinical development of adaphostin as a novel agent for the treatment of CLL appears warranted.

AbstractWe recently reported that chronic lymphocytic leukemia (CLL) cells synthesize and release vascular endothelial growth factor (VEGF) under normoxic and hypoxic conditions. CLL B cells also express VEGF membrane receptors (VEGF-R1 and VEGF-R2), suggesting that they use VEGF as a survival factor. To assess the mechanism of apoptosis resistance related to VEGF, we determined the impact of VEGF on CLL B cells, and we studied the impact of epigallocatechin-3-gallate (EGCG), a known receptor tyrosine kinase (RTK) inhibitor, on VEGF receptor status and viability of CLL B cells. VEGF165 significantly increased apoptotic resistance of CLL B cells, and immunoblotting revealed that VEGF-R1 and VEGF-R2 are spontaneously phosphorylated on CLL B cells. EGCG significantly increased apoptosis/cell death in 8 of 10 CLL samples measured by annexin V/propidium iodide (PI) staining. The increase in annexin V/PI staining was accompanied by caspase-3 activation and poly–adenosine diphosphate ribose polymerase (PARP) cleavage at low concentrations of EGCG (3 μg/mL). Moreover, EGCG suppressed the proteins B-cell leukemia/lymphoma-2 protein (Bcl-2), X-linked inhibitor of apoptosis protein (XIAP), and myeloid cell leukemia-1 (Mcl-1) in CLL B cells. Finally, EGCG (3-25 μg/mL) suppressed VEGF-R1 and VEGF-R2 phosphorylation, albeit incompletely. Thus, these results suggest that VEGF signaling regulates survival signals in CLL cells and that interruption of this autocrine pathway results in caspase activation and subsequent leukemic cell death.