ALBERT

Description: Vascular endothelial cell growth factor (VEGF) is a multifunctional cytokine involved in tumor formation. In chronic lymphocytic leukemia (CLL), it is known that the malignant cells secrete VEGF and possess VEGF receptors. This suggests that an autocrine loop might be important in the pathogenesis of CLL. Here we show that, in patients with lymphadenopathy, autocrine VEGF and α4β1 integrin are involved in the chemokine-dependent motility of CLL cells on and through endothelium—processes important for the invasion of lymphoreticular tissues, a major determinant of disease outcome. In contrast, normal lymphocytes were not dependent on autocrine VEGF or α4β1 for either type of cell movement. Moreover, in contrast to normal B lymphocytes, CLL cells failed to cluster and activate αLβ2 in response to chemokines, unless VEGF receptor(s) and α4β1 were also engaged by their respective ligands. This is the first demonstration that autocrine VEGF is involved in CLL-cell motility, and that the αLβ2 on the malignant cells is functionally altered compared with that of normal B cells in not undergoing activation in response to chemokine alone. Given the importance of cell motility for tissue invasion, the present results provide a rationale for a trial of VEGF and α4 blockade in patients with CLL who have tissue disease. (Blood. 2005;105:4813-4819)

Description: Tyrosine kinase inhibitors (TKIs) that preferentially inhibit the oncogenic Bcr-Abl fusion protein have radically changed the treatment of chronic myeloid leukaemia (CML). Imatinib mesylate ((IM) Gleevec®, Glivec®, STI571; Novartis) has been shown to induce almost complete hematological responses in patients after 12 months and major cytogenetic responses in 69%. However, the proportion of patients achieving molecular responses or having undetectable Bcr-Abl transcripts remains low. In addition to this failure to induce molecular response, resistance occurs in 10–15% of patients. Nilotinib ((NIL) AMN107; Novartis) has approximately 20-fold greater potency in in vitro assays but despite this improved potency in vitro and on bulk primary CML cells we have found that NIL is no better than IM in inducing apoptosis of the primitive CD34+ CML cell population. We previously showed that as with IM, CD34+ CML cells persist after NIL treatment and that NIL also has an anti-proliferative rather than pro-apoptotic effect, resulting in the accumulation of quiescent CD34+ cells. It has been proposed that this population of TKI-insensitive primitive cells may form a pool of disease in patients under treatment and contribute to the Bcr-Abl molecular signal detected in the majority of patients. This population must therefore, be specifically targeted in order to eradicate the disease in patients and to result in cure rather than control of the disease. One possible reason for the failure of NIL to kill CD34+ CML cells is that the cells do not accumulate sufficient intracellular levels of the drug due to either inadequate active uptake via the SLC transporter family, or to efflux via multidrug resistance proteins of the ATP-binding cassette (ABC) family. In order to determine the interaction of NIL with major clinically implicated drug transporters we have investigated interactions with ABCB1, MRP1 (ABCC1), ABCG2 and hOCT1 in CML cell lines and primitive (CD34+) primary CML cells. Determination of the distribution coefficient (logD) showed that NIL is more lipophilic than IM (logD of 2.4 vs 0.8 respectively) and assays using 14C-NIL in OCT1 over expressing KCL22 CML cells showed that accumulation of NIL is neither temperature dependent nor reduced by the inhibitors prazosin or amatadine. These data demonstrate that NIL uptake is not dependent on active import by hOCT1, however NIL did inhibit the uptake of a known OCT1 substrate tetraethylammonium bromide (TEA). Efflux assay in cell lines over-expressing MDR1, MRP1 or ABCG2 showed that NIL is not effluxed by any of these proteins. Over expression &/or the addition of specific inhibitors (PSC833, MK571, fumitremorgin C respectively) did not alter the cellular accumulation of NIL as assayed by radiolabelled drug accumulation assays, transepithelial transport, HPLC analysis or when p-CrkL was measured as a surrogate assay for Bcr-Abl inhibition. Using efflux assays with known substrates we found that NIL inhibited rhodamine efflux via MDR1, or BODIPY-prazosin efflux via ABCG2, but had no effect on Fluo-3 efflux via MRP1. Thus, rather than being a substrate NIL acts as an inhibitor of MDR1 and ABCG2 in these assays. To conclusively assess the effect of transporter activity in primary cells we first measured the expression of the transporters in primitive CD34+ CML cells. Unlike OCT1, the efflux transporters MDR1, MRP1 and ABCG2 are all expressed on CML CD34+ cells. When compared to normal CD34+ cells ABCG2 was over expressed (291%), MRP1 was expressed at very similar levels (108%) and surprisingly MDR1 was expressed at a much lower level in CML (13.5%). We treated CD34+ CML cells with NIL for 72hrs in the presence or absence of the transporter inhibitors. NIL reduced the total cell number and p-CrkL activity as expected however, these effects were not potentiated by any of the transporter inhibitors. Furthermore, NIL increased the number of quiescent CD34+ cells remaining after treatment but again transporter inhibitors did not modulate this effect. Therefore, we have found no evidence for either active uptake of NIL via hOCT1 or efflux via MDR1, MRP1 or ABCG2. It is unlikely that these transporters will have any effect on the clinical response to this drug in either MNCs or the more resistant CD34+ stem cell population.

Description: We have previously shown that imatinib is a substrate for both the efflux transporter P-glycoprotein (ABCB1; MDR-1 gene product) and the influx transporter human Organic Cation Transporter 1 (hOCT1; SLCA22). High hOCT1 and low ABCB1 expression levels in chronic myeloid leukaemia (CML) patients correlate with improved clinical outcome (Wang L. et al. Clinical Pharmacology and Therapeutics; epub June 13th 2007). The second generation tyrosine kinase inhibitor nilotinib is 30-fold more potent than imatinib and is effective in imatinib-resistant patients. However, its cellular transport has not been characterised. The CML cell line KCL22 was used as it has low basal hOCT1 expression. Drug transport studies of 14C-radiolabelled nilotinib (kind gift from Novartis) were performed using high expressing hOCT1 transfected KCL22 cells, with or without OCT1 and ABCB1 inhibitors. Further vectorial ABCB1 efflux studies were performed on confluent monolayers of ABCB1 transfected Type II Madin Darby canine kidney (MDCKII) cells. Nilotinib efflux was not modulated in the presence of the ABCB1 inhibitors PSC-833 (10μM), tariquidar (500nM) and verapamil (500μM), and no evidence of transport via ABCB1 was seen in the transfected ABCB1 MDCKII cells. Nilotinib influx was not modulated in hOCT1 over-expressing cells, or in the presence of OCT1 inhibitors prazosin (100μM) and amantadine (500μM). Nilotinib uptake was also not modulated by the hOCT1 substrate tetraethylamine (TEA) (5μM), however nilotinib inhibited TEA uptake. The distribution coefficient (logD) of nilotinib was 2.1, compared with 0.81 for imatinib, demonstrating high lipophilicity for nilotinib. Flow cytometry revealed greater suppression of phospho-CrKL (a surrogate marker for BCR-ABL blockade) with nilotinib after 1 hour when compared with imatinib, regardless of hOCT1 expression, indicating that nilotinib is effective at BCR-ABL suppression even at low hOCT1 levels, in contrast to imatinib. Nilotinib was not a substrate for the ABCB1 and hOCT1 transporters; however it may be a potential inhibitor of hOCT1. Therefore the level of expression of ABCB1 and hOCT1 in CML is unlikely to be an indicator of the clinical outcome for patients receiving nilotinib, in contrast to what we have shown for imatinib. Nilotinib may therefore be effective in patients who have low hOCT1 expression before start of therapy.

Description: A sequence of 31 amino acids (S565-K595) in domain 6 of the light chain of high molecular weight kininogen (HK) has previously been shown to be responsible for the binding of plasma prekallikrein (PK) or kallikrein. To find effective peptides that might block binding between HK and PK on cell surfaces, a new series of synthetic peptides has now been prepared that incorporates portions of this binding domain sequence. For mapping the minimal sequence within HK, these new peptides were tested for their ability to compete with HK for binding PK in a cell-free system and on human umbilical vein endothelial cells (HUVEC). In the former, at pH 7.4, the kds for binding between kallikrein and either D567-K595, S565-P594, D567-S593, or D567-T591 were all similar to that for the binding of S565-K595 (0.2 to 0.4 μmol/L), but those for the binding of D568-K595, W569-K595, and D567-P589 were an order of magnitude greater (kd = 2 to 5 μmol/L). D567-S586, the shortest chain length of the N- and C-terminal truncation sequences tested, does not effectively compete with kininogen for kallikrein binding (kd = 100 μmol/L). These results imply that D567-T591, a 25-residue peptide (HK25c), contains sufficient structural information for binding kallikrein in solution. D567-T591 also is the minimum structural sequence to block binding of kallikrein to HUVEC-bound HK (IC50 = 50 nmol/L) and to inhibit PK activation to kallikrein on the cell surface (IC50 = 80 nmol/L). In addition, D567-T591 also inhibits the generation of kallikrein-activated urokinase, which activates plasminogen to plasmin (IC50 = 100 nmol/L). Thus, HK-derived peptides may be useful compounds for modulating excessive fibrinolysis and hypotension in sepsis and multiple trauma.

Description: Prospective identification of patients whose chronic myeloid leukemia (CML) will progress to blast crisis is currently not possible. PP2A is a phosphatase and tumor suppressor that regulates cell proliferation, differentiation, and survival. Cancerous inhibitor of PP2A (CIP2A) is a recently described inhibitor of PP2A in breast and gastric cancer. The aim of this study was to investigate whether CIP2A played a role in CML and whether PP2A or its inhibitor proteins CIP2A or SET could predict clinical outcome. At the time of diagnosis of CML, patients who will later progress to blast crisis have significantly higher levels of CIP2A protein (P 〈 .0001) than patients who do not progress, suggesting that PP2A is functionally inactive. We show that the potential mechanism for disease progression is via altered phosphorylation of the oncogene c-Myc. Knockdown of CIP2A results in increased PP2A activity, decreased c-Myc levels, and a decrease in BCR-ABL1 tyrosine kinase activity. We demonstrate that CIP2A levels at diagnosis can consistently predict patients who will progress to blast crisis. The data show that CIP2A is biologically and clinically important in CML and may be a novel therapeutic target.