Increased serum level of alpha-2 macroglobulin and its production by B-lymphocytes in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL), the most common adult's leukemia in the western world, is caused in 95% of the cases by uncontrolled proliferation of monoclonal B-lymphocytes. The complement system in CLL is chronically activated at a low level via the classical pathway (CP). This chronic activation is induced by IgG-hexamers, which are formed after binding to alpha-2-macroglobulin (A2M). The study investigated for the first time the serum levels of A2M in CLL patients, their association with the disease severity, and A2M production by the malignant B-lymphocytes. Blood samples were collected from 65 CLL patients and 30 normal controls (NC) subjects, and used for quantifications of the A2M levels, the complement activation marker (sC5b-9), the complement components C2, C3 and C4, and clinical biochemistry and hematology parameters. The production of A2M was studied in B-lymphocytes isolated from blood samples as well as in CLL and non-CLL cell lines.The serum A2M levels were significantly higher in CLL patients vs NCs, showing values of 3.62 ± 0.22 and 1.97 ± 0.10 mg/ml, respectively. Within the CLL group, A2M levels correlated significantly with the disease stage, with sC5b-9, and with clinical indicators of the disease severity. Increased A2M production was showed in three out of four CLL B-lymphocytic lines that were studied, as compared to non-CLL lines, to a non-lymphocytic line, and to blood-derived primary B-lymphocytes. A2M production was further increased both in primary cells and in the CLL cell-line after incubation with CLL sera, compared to NC sera. This study shows for the first time that serum A2M levels in CLL are significantly increased, likely due to A2M production by the malignant B-lymphocytes, and are correlated with the disease severity and with chronic complement activation. The moderate change in A2M production after incubation with NC sera in-vitro supports the hypothesis that inhibition of excess A2M production can be achieved, and that this may potentially down-regulate the IgG-hexamerization and the resulting chronic CP activation. This may also help restore complement system activity, and eventually improve complement activity and immunotherapy outcomes in CLL.

Inhibiting mutant KRAS G12D gene expression using novel peptide nucleic acid-based antisense: A potential new drug candidate for pancreatic cancer.

KRAS mutations, which are the main cause of the pathogenesis of lethal pancreatic adenocarcinomas, impair the functioning of the GTPase subunit, thus rendering it constitutively active and signaling intracellular pathways that end with cell transformation. In the present study, the AsPC-1 cell line, which has a G12D-mutated KRAS gene sequence, was utilized as a cellular model to test peptide nucleic acid-based antisense technology. The use of peptide nucleic acids (PNAs) that are built to exhibit improved hybridization specificity and have an affinity for complementary RNA and DNA sequences, as well as a simple chemical structure and high biological stability that affords resistance to nucleases and proteases, enabled targeting of the KRAS-mutated gene to inhibit its expression at the translation level. Because PNA-based antisense molecules should be capable of binding to KRAS mRNA sequences, PNAs were utilized to target the mRNA of the mutated KRAS gene, a strategy that could lead to the development of a novel drug for pancreatic cancer. Moreover, it was demonstrated that introducing new PNA to cells inhibited the growth of cancer cells and induced apoptotic death and, notably, that it can inhibit G12D-mutated KRAS gene expression, as demonstrated by RT-PCR and western blotting. Altogether, these data strongly suggest that the use of PNA-based antisense agents is an attractive therapeutic approach to treating KRAS-driven cancers and may lead to the development of novel drugs that target the expression of other mutated genes.