Investigating residual leukemic cells in acute lymphoblastic leukemia: a practical approach using a streamlined interphase fluorescence in situ hybridization method on cerebrospinal fluid.

INTRODUCTION: A precise diagnosis of central nervous system involvement in acute lymphoblastic leukemia (ALL) requires comprehensive knowledge of morphological analysis, with a focus on the quantity and quality of cells being examined. Some research has utilized techniques such as immunocytochemistry, flow cytometry, polymerase chain reaction (PCR), and interphase fluorescence in situ hybridization (iFISH) on cerebrospinal fluid (CSF) cytospin samples to detect any remaining leukemic cells in the CSF. To obtain reliable results using immunocytochemistry and flow cytometry, it is essential to use freshly collected specimens within a limited timeframe. At the same time, PCR requires a sufficient number of cells for DNA extraction. On the other hand, the iFISH procedure on CSF cytospin samples can be challenging and requires practice. Therefore, there is a need for a fast, easy method that will be affordable and marketable in laboratories where the above methods are not available, or the sample is insufficient to use those methods. METHODS: The samples were prepared by centrifugation of 1 mL aliquots of CSF collected into EDTA tubes. The CSF sample was centrifuged at 3000 rpm for 3 min, the supernatant was removed, and the pellet was placed in KCl hypotonic solution for 5 min at 37 °C. Other steps (fixation, hybridization, wash steps, and analysis) were the same as in the standard protocol for blood samples. The BCR-ABL1 rearrangements were performed and evaluated in 200 interphase cells. RESULTS: 90% of Ph(+) cells were found in CSF. CONCLUSION: We propose a significantly streamlined iFISH method for detecting blast/residual leukemic cells in acute lymphoblastic leukemia using CSF as a complementary test option.

Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de novo pyrimidine biosynthesis inhibition.

There is an increasing interest in the field of cancer therapy for small compounds targeting pyrimidine biosynthesis, and in particular dihydroorotate dehydrogenase (DHODH), the fourth enzyme of this metabolic pathway. Three available DHODH structures, featuring three different known inhibitors, were used as templates to screen in silico an original chemical library from Erevan University. This process led to the identification of P1788, a compound chemically related to the alkaloid cerpegin, as a new class of pyrimidine biosynthesis inhibitors. In line with previous reports, we investigated the effect of P1788 on the cellular innate immune response. Here we show that pyrimidine depletion by P1788 amplifies cellular response to both type-I and type II interferons, but also induces DNA damage as assessed by γH2AX staining. Moreover, the addition of inhibitors of the DNA damage response led to the suppression of the P1788 stimulatory effects on the interferon pathway. This demonstrates that components of the DNA damage response are bridging the inhibition of pyrimidine biosynthesis by P1788 to the interferon signaling pathway. Altogether, these results provide new insights on the mode of action of novel pyrimidine biosynthesis inhibitors and their development for cancer therapies.

Phenoxypropanolamine derivatives as selective inhibitors of the 20S proteasome ß1 and ß5 subunits.

New series of thiophene-containing phenoxypropanolamines were synthesized and evaluated for their potency to inhibit the three proteolytic activities of the mammalian 20S proteasome. Noticeable inhibition of both ChT-L and PA activities was obtained with three compounds: one with unsubstituted phenoxypropanolamine group (7) and the two others with a p-Cl-substituted group (4 and 9). For three other compounds (3, 8 and 10), ChT-L activity alone was significantly inhibited. In silico docking performed on the ß5 and ß1 subunits bearing the respective ChT-L and PA catalytic sites showed features common to poses associated with active compounds. These features may constitute a selectivity criterion for structure-guided inhibitor design.

New C(4)- and C(1)-derivatives of furo[3,4-c]pyridine-3-ones and related compounds: evidence for site-specific inhibition of the constitutive proteasome and its immunoisoform.

A set of 18 new C(4) and C(1) derivatives of nor-cerpegin (1,1-dimethyl furo[3,4-c]pyridine-3-one), 6 model compounds (γ- and δ-lactones) and 20 furo- or thieno[2,3-d]-pyrimidine-4-one related compounds were designed and synthesized. Each compound was assayed for inhibition of CT-L, T-L and PA proteolytic activities of 20S constitutive proteasome (c20S). Most performant compounds were also assayed on 20S immunoproteasome (i20S). Compound 10 with a benzylamino group at C(4) and dimethylated at C(1) of the furopyridine ring was the most efficient PA site-specific inhibitor of the c20S (IC50(cPA) of 600nM) without noticeable inhibition of the i20S PA site (iPA). In silico docking assays for 10 at the iPA catalytic site revealed the absence of poses normally observed for this compound and related ones at the constitutive PA site (cPA). The thieno[2,3-d]pyrimidine-4-one 40 was T-L site-specific with a mild inhibition of both c20S and i20S in vitro (IC50(cT-L) of 9.9µM and IC50(iT-L) of 6.7µM). In silico docking assays of 40 at T-L sites of c20S and i20S revealed almost identical first rank poses in the two types of sites with no possibility left for nucleophilic attack by Thr1 as observed for the fused furopyridine-3-one 10.

C1 and N5 derivatives of cerpegin: synthesis of a new series based on structure-activity relationships to optimize their inhibitory effect on 20S proteasome.

Thirty-two new derivatives of cerpegin (1,1,5-trimethylfuro[3,4-c]pyridine-3,4-dione) were designed and synthesized in high yield by a new method, combining several C(1) and N(5) substituents. All compounds were tested for their inhibitory effect on the CT-L, T-L and PA proteolytic activities of a purified mammalian 20S proteasome. Only one molecule inhibited both CT-L and PA activities. Sixteen molecules specifically inhibited PA at the micromolar range, out of which fourteen had IC50 values around 5 µM and two had IC50 values closer to 2 µM. Except in one case, neither calpain I nor cathepsin B was inhibited. In silico docking suggests a unique mode of binding of the most efficient compounds to the ß1 catalytic site (PA activity) in relation to the chemical nature of C(1) substituents.

A new series of N5 derivatives of the 1,1,5-trimethyl furo[3,4-c]pyridine-3,4-dione (cerpegin) selectively inhibits the post-acid activity of mammalian 20S proteasomes.

A large set of N(5)-derivatives of cerpegin (1,1,5-trimethyl furo[3,4-c]pyridine-3,4-dione) was designed and synthesized in high yields by a simple and handy method using various primary amines for a pyridine cycle synthesis. The effects of 29 derivatives on the three types of catalytic sites of purified mammalian 20S proteasomes (CT-L, T-L and PA) were measured. Most of the new compounds specifically inhibited the PA activity, in the micromolar range. Docking experiments support these results. Moreover, neither calpain I nor cathepsin B were inhibited.

Novel complex t(V;9;22) rearrangements in three cases with chronic myeloid leukemia and a rare translocation in a case with classical Philadelphia chromosome.

The fusion gene BCR/ABL arises in connection with a complex translocation event in 2-10% of cases with chronic myeloid leukemia (CML). Due to causative treatment with Imatinib most cases with variant rearrangements show no specific prognostic significance, though the events of therapy resistance remain to be studied. Herein we report on three CML cases with complex chromosomal aberrations not observed before, involving chromosomal regions such as 1p32, 2q11 and 6q12. Additionally we report on one case with the rare translocation t(3;8)(p22;q22) along with the classic Philadelphia (Ph) chromosome. In two cases, two different breakpoints on chromosome 22 were found. Moreover, in one of them two breakpoints on chromosome 9 were observed. The following chromosomal studies, during therapy by Imatinib, have revealed different cytogenetic responses.